Methods and compositions for treating and/or preventing a disease or disorder associated with abnormal level and/or activity of the ifp35 family of proteins

ABSTRACT

The present invention relates to methods and compositions for treating and/or preventing a disease or disorder associated with abnormally high level of the IFP35 family of proteins, including IFP35 and NMI, methods and compositions for diagnosis, prognosis or treatment monitoring of a disease or disorder associated with abnormally high level of the IFP35 family of proteins, including IFP35 and NMI, and methods and compositions for identifying a modulator of the IFP35 family of proteins, including IFP35 and NMI.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to Chinese patent applicationNo. 201410418773.1, filed Aug. 22, 2014, the disclosure of which isincorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

In some aspects, the present disclosure relates to methods andcompositions for treating and/or preventing a disease or disorderassociated with abnormally level and/or activity of the IFP35 family ofproteins, including IFP35 and NMI; methods and compositions fordiagnosis, prognosis or treatment monitoring of a disease or disorderassociated with abnormally level and/or activity of the IFP35 family ofproteins; and methods and compositions for identifying a modulator ofthe IFP35 family of proteins.

BACKGROUND ART

Endogenous danger signals are “damage-associated molecular patterns”(DAMPs) that are released from necrotic or stressed cells which triggerthe inflammatory response. For instance, cells produce endogenous DAMPsfor immune regulation after infection or injury. See Gallucci et al.,Current Opinion in Immunology 13, 114-119 (2001); Matzinger, Science296, 301-305 (2002); and Hirsiger et al., Mediators of Inflammation,315941 (2012). The known DAMPs include not only small molecules such asuric acid, cholesterol and ATP (see Gombault et al., Frontiers inImmunology 3, 414 (2012); Heil et al., Frontiers in Plant Science 5, 578(2014); and Shi, Journal of Clinical Investigation 120, 1809-1811(2010)), but also proteins such as high-mobility group box protein 1(HMGB1) (see Tsung et al., Journal of Internal Medicine 276, 425-443(2014); and Wang et al., Science 285, 248-251 (1999)), heat shockproteins (HSPs) (see Tamura et al., Immunotherapy 4, 841-852 (2012)),interleukin-1α (IL-1α) (see Dinarello, Annual Review of Immunology 27,519-550 (2009)), interleukin-33 (IL-33) (see Liew et al., Nature ReviewsImmunology 10, 103-110 (2010)), myeloid-related protein-8 (Mrp8) andmyeloid-related protein-14 (Mrp14) (see Vogl et al., Nature Medicine 13,1042-1049 (2007); and Austermann et al., Cell Reports 9, 2112-2123(2014)). DAMPs may play important roles in pathogen elimination anddamage repair. However, uncontrolled activation of inflammation by DAMPswill lead to persistent over-expression of toxic cytokines, which aremajor risk factors for sepsis shock and systemic inflammatory responsesyndrome. See Srikrishna et al., Neoplasia 11, 615-628 (2009); andPiccinini et al., Mediators of Inflammation, doi:10.1155/2010/672395(2010).

Thousands of cytokines are involved in the immune response. Beingselectively activated or restrained, they constitute a complicate butprecisely under-controlled network to perform antiviral or immuneregulation functions. See Dinarello, Chest 118, 503-508 (2000). Inaddition, they are considered to be broadly involved in other cellularactivities, for instance, suppressing tumor growth, promotingcatabiosis, and inducing apoptosis. See Crusz et al., Nature ReviewsClinical Oncology, doi:10.1038/nrclinonc.2015.105 (2015); Chen et al.,Drug Design, Development and Therapy 9, 2941-2946 (2015); Villeda etal., Nature 477, 90-94 (2011); and Eckhardt et al., Cell Death Disease5, e1562 (2014). However, the functional mechanisms of these proteinsare largely unclear.

SUMMARY

In one aspect, disclosed herein is a method for treating and/orpreventing a disease or disorder associated with abnormally high leveland/or activity of IFP35 (Interferon-induced Protein 35 kD) and/or NMI(N-Myc-interacting protein) in a subject. In some embodiments, themethod comprises administering, to a subject in need of such treatmentand/or prevention, an effective amount of an agent that prevents orreduces production and/or an activity of IFP35 and/or NMI in thesubject.

In any of the preceding embodiments, the disease or disorder associatedwith abnormally high level and/or activity of IFP35 and/or NMI can beassociated with excessive immune response. In any of the precedingembodiments, the disease or disorder can be associated with cytokinestorm. In any of the preceding embodiments, the disease or disorder canbe selected from the group consisting of inflammation, infection, organdamage, sepsis, and autoimmune disease. In any of the precedingembodiments, the method can be for treating a disease or disorderassociated with abnormally high level and/or activity of IFP35 and/orNMI. In any of the preceding embodiments, the method can be forpreventing a disease or disorder associated with abnormally high leveland/or activity of IFP35 and/or NMI.

In any of the preceding embodiments, the agent can comprise a molecule,such as a small molecule or a polypeptide, that inhibits or reduces theexpression and/or activity of IFP35 and/or NMI. In any of the precedingembodiments, the agent can comprise an antibody or antigen bindingfragment thereof that inhibits or reduces the expression and/or activityof IFP35 and/or NMI. In any of the preceding embodiments, the agent cancomprise a polynucleotide, such as an siRNA, shRNA, or miRNA, thattargets the gene encoding IFP35 and/or NMI. In any of the precedingembodiments, the agent can comprise an antisense polynucleotide, such asan antisense RNA, that targets the gene encoding IFP35 and/or NMI. Inany of the preceding embodiments, the agent can comprise a molecule thatinhibits or reduces the oligomerization of IFP35 and/or NMI. In any ofthe preceding embodiments, the agent can comprise a molecule thatinhibits or reduces the expression and/or activity of an interferon,thereby inhibiting or reducing the expression and/or activity of IFP35and/or NMI. In any of the preceding embodiments, the agent can comprisea molecule that inhibits or reduces the secretion of IFP35 and/or NMI.In any of the preceding embodiments, the agent can comprise a moleculethat inhibits or reduces the interaction between IFP35 and/or NMI and acellular receptor of IFP35 and/or NMI. In any of the precedingembodiments, the agent can comprise a molecule that inhibits or reducesthe interaction between IFP35 and/or NMI and a IFP35 and/or NMI cellsurface receptor.

In any of the preceding embodiments, the prevention or reduction of theproduction and/or the activity of IFP35 and/or NMI can result in theinhibition or reduction of the expression and/or activity of aninflammatory factor, and/or inhibiting or reduction of NF-κB signaling.In one aspect, the inflammatory factor comprises IL-1β, TNF-α, iNOS,and/or CD86, and the NF-κB signaling is mediate by a TLR such as TLR4.

In any of the preceding embodiments, the agent can comprise an antibodyor antigen binding fragment that specifically binds to IFP35 and/or anantibody or antigen binding fragment that specifically binds to NMI. Inone aspect, the antibody or antigen binding fragment specifically bindsone or more NIDs (NMI/IFP35 domains).

In any of the preceding embodiments, the method can further compriseadministering a pharmaceutically acceptable carrier or excipient. In anyof the preceding embodiments, the agent can be administered via an oral,nasal, inhalational, parental, intravenous, intraperitoneal,subcutaneous, intramuscular, intradermal, topical, or rectal route. Inany of the preceding embodiments, the agent can be administered at anamount that reduces production and/or an activity of IFP35 and/or NMI inthe subject to a level that is substantially identical to a productionand/or an activity level of IFP35 and/or NMI in a comparable subjectthat does not have a disease or disorder associated with abnormally highlevel and/or activity of IFP35 and/or NMI.

In any of the preceding embodiments, the subject can be a mammal. Insome embodiments, the mammal is a human or a non-human mammal.

In another aspect, disclosed herein is a pharmaceutical composition fortreating and/or preventing a disease or disorder associated withabnormally high level and/or activity of IFP35 and/or NMI in a subject.In some aspects, the pharmaceutical composition comprises an effectiveamount of an agent that prevents or reduces production and/or anactivity of IFP35 and/or NMI in a subject and a pharmaceuticallyacceptable carrier or excipient. In any of the preceding embodiments,the pharmaceutical composition can further comprise an effective amountof a drug for the treatment and/or prevention of a disease or disorderassociated with abnormally high level and/or activity of IFP35 and/orNMI in the subject.

In yet another aspect, disclosed herein is use of an effective amount ofan agent that prevents or reduces production and/or an activity of IFP35and/or NMI in a subject for the manufacture of a medicament for treatingand/or preventing a disease or disorder associated with abnormally highlevel and/or activity of IFP35 and/or NMI in the subject. In someaspects, the disease or disorder associated with abnormally high leveland/or activity of IFP35 and/or NMI is inflammation, infection, sepsis,organ damage, and autoimmune disease.

In still another aspect, disclosed herein is an antibody or antigenbinding fragment thereof that specifically binds to IFP35 and/or NMI. Insome aspects, the antibody or antigen binding fragment specificallybinds to an epitope within SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, orSEQ ID NO: 8. In some embodiments, the antibody or antigen bindingfragment specifically binds to an epitope within amino acids 81-170,177-268, or 136-216 of SEQ ID NO: 2. In other embodiments, the antibodyor antigen binding fragment specifically binds to an epitope withinamino acids 81-168, 175-266, or 134-214 of SEQ ID NO: 4. In still otherembodiments, the antibody or antigen binding fragment specifically bindsto an epitope within amino acids 104-193, 202-293, or 151-250 of SEQ IDNO: 6. In other embodiments, the antibody or antigen binding fragmentspecifically binds to an epitope within amino acids 103-192, 201-292, or151-240 of SEQ ID NO: 8.

In another aspect, disclosed herein is an antibody or antigen bindingfragment thereof that specifically binds to IFP35 and/or NMI, and theantibody or antigen binding fragment thereof comprises a heavy chainvariable region comprising a complementarity determining region (CDR)consisting of the amino acid sequences of a CDR in the heavy chainvariable region sequence set forth in SEQ ID NO: 9 and/or a light chainvariable region comprising a CDR consisting of the amino acid sequenceof a CDR in the light chain variable region sequence set forth in SEQ IDNO: 10. In one embodiment, the antibody or antigen binding fragmentthereof comprises a heavy chain variable region set forth in SEQ ID NO:9 and a light chain variable region set forth in SEQ ID NO:10.

In any of the preceding embodiments, the antibody can be a monoclonalantibody, a polyclonal antibody, or a bi-specific antibody. In any ofthe preceding embodiments, the antigen binding fragment can be an Fab,F(ab □)2, Fv or scFv fragment. In any of the preceding embodiments, theantibody or antigen binding fragment can further comprise a human heavychain constant region and a human light chain constant region.

In any of the preceding embodiments, the antibody or antigen bindingfragment can be a fully human antibody or antigen binding fragment or ahumanized antibody or antigen binding fragment. In any of the precedingembodiments, the antibody or antigen binding fragment can berecombinantly produced.

In one aspect, disclosed herein is a method for treating and/orpreventing a disease or disorder associated with abnormally high leveland/or activity and/or activity of IFP35 and/or NMI in a subject. Themethod can comprise administering, to a subject in need of suchtreatment and/or prevention, an effective amount of the antibody orantigen binding fragment according to any of the preceding embodiments.In any of the preceding embodiments, the disease or disorder can beassociated with excessive immune response. In any of the precedingembodiments, the disease or disorder can be associated with cytokinestorm. In any of the preceding embodiments, the disease or disorderassociated with abnormally high level and/or activity of IFP35 and/orNMI can be selected from the group consisting of inflammation,infection, organ damage, sepsis, and autoimmune disease. In any of thepreceding embodiments, the prevention or reduction of the productionand/or the activity of IFP35 and/or NMI can result in the inhibition orreduction of the expression and/or activity of an inflammatory factor,and/or inhibiting or reduction of NF-κB signaling. In one aspect, theinflammatory factor comprises IL-1β, TNF-α, iNOS, and/or CD86, and theNF-κB signaling is mediate by a TLR such as TLR4.

In one aspect, disclosed herein is an isolated polynucleotide encodingan antibody or antigen binding fragment thereof that specifically bindsto IFP35 and/or NMI. In some embodiments, the antibody or antigenbinding fragment thereof comprises a heavy chain variable regioncomprising a complementarity determining region (CDR) consisting of theamino acid sequence of a CDR in the heavy chain variable region sequenceset forth in SEQ ID NO: 9 and/or a light chain variable regioncomprising a CDR consisting of the amino acid sequences of a CDR in thelight chain variable region sequence set forth in SEQ ID NO: 10. In someaspects, the antibody or antigen binding fragment thereof comprises aheavy chain variable region set forth in SEQ ID NO: 9 and a light chainvariable region set forth in SEQ ID NO:10.

In one aspect, also disclosed herein is an isolated vector comprisingthe polynucleotide according to any of the preceding embodiments. Inanother aspect, also disclosed herein is an isolated host cellcomprising the vector according to any of the preceding embodiments.

In another aspect, disclosed herein is a host cell which is selectedfrom the group consisting of the following: (a) a host cell transformedwith an expression vector comprising a polynucleotide comprising asequence encoding a heavy chain variable region of an antibody orantigen binding fragment thereof and a polynucleotide comprising asequence encoding a light chain variable region of an antibody orantigen binding fragment thereof; and (b) a host cell transformed withan expression vector comprising a polynucleotide comprising a sequenceencoding a heavy chain variable region of an antibody or antigen bindingfragment thereof and an expression vector comprising a polynucleotidecomprising a sequence encoding a light chain variable region of anantibody or antigen binding fragment thereof. In some embodiments, theantibody or fragment comprises a heavy chain variable region comprisinga complementarity determining region (CDR) consisting of the amino acidsequences of a CDR in the heavy chain variable region sequence set forthin SEQ ID NO: 9 and/or a light chain variable region comprising a CDRconsisting of the amino acid sequences of a CDR in the light chainvariable region sequence set forth in SEQ ID NO: 10. In any of thepreceding embodiments, the host cell can be a eukaryotic cell such as aCHO cell.

In another aspect, disclosed herein is a method of making an anti-IFP35antibody or antigen binding fragment thereof and/or an anti-NMI antibodyor antigen binding fragment thereof. In some aspects, the methodcomprises: a) culturing the host cell of any one of precedingembodiments under conditions suitable for expression of thepolynucleotide encoding the antibody or antigen binding fragment; and b)isolating the antibody or antigen binding fragment. In one aspect, alsodisclosed herein is an antibody or antigen binding fragment produced bythe method according to any of the preceding embodiments.

In some embodiments, disclosed herein is an isolated polypeptidecomprising, consisting essentially of, or consisting of: (1) thesequence set forth in amino acids 81-170, 177-268, or 136-216 of SEQ IDNO: 2; or (2) the sequence set forth in amino acids 81-168, 175-266, or134-214 of SEQ ID NO: 4; or (3) the sequence set forth in amino acids104-193, 202-293 or 151-250 of SEQ ID NO: 6; or (4) the sequence setforth in amino acids 103-192, 201-292, or 151-240 of SEQ ID NO: 8.

In some embodiments, disclosed herein is an isolated polypeptidecomprising, consisting essentially of, or consisting of the sequence setforth in amino acids 81-170, 177-268, or 136-216 of SEQ ID NO: 2,wherein one or more of the amino acid residues at positions 145, 147,150, 151, 172, 173, 175, 177, 182, 188, 192, 212, 199, 201, 207, 208,210, 214, and 216 of SEQ ID NO: 2 (Ser145, Arg147, Glu150, Glu151,Asp172, Val173, Glu175, Leu177, Met182, Asp188, Gln192, Arg212, Gln199,Thr201, Gln207, Gln208, Pro210, Ser214, and Tyr216) are mutated and/ormodified. In some aspects, according to structure-based sequencealignment, the left NID domain outside of the determined IFP35 NID-Hdomain structure has similar structure to NID-H. Ser234, Leu236, Arg110,Arg 108, Arg233, Leu 82, Glu268, Thr271, Val273, Glu92, Asp87, Asp245,and Gly246 of SEQ ID NO: 2 are the amino acid residues in the left NIDdomain that correspond to the surface residues in the determined NIDdomain structure of IFP35. In any of the preceding embodiments, themutation(s) can substantially reduce or eliminate binding of thepolypeptide to a IFP35 receptor or an inhibitory antibody specific forIFP35.

In some embodiments, disclosed herein is an isolated polypeptidecomprising, consisting essentially of, or consisting of the sequence setforth in amino acids 81-168, 175-266, or 134-214 of SEQ ID NO: 4,wherein one or more of the amino acid residues at positions 143, 145,148, 149, 170, 172, 173, 175, 180, 186, 190, 210, 197, 199, 204, 205,206, 208, 212, and 214 of SEQ ID NO: 4 (Ser143, Arg145, Glu148, Glu149,Glu170, Arg172, Glu173, Leu175, Met180, Glu186, Gln190, Arg210, Gln197,Arg199, Arg204, Gln205, Gln206, Leu208, Ser212, and Tyr214) are mutatedand/or modified. In any of the preceding embodiments, the mutation(s)can substantially reduce or eliminate binding of the polypeptide to aIFP35 receptor or an inhibitory antibody specific for IFP35.

In some embodiments, disclosed herein is an isolated polypeptidecomprising, consisting essentially of, or consisting of the sequence setforth in amino acids 104-193, 202-293, or 151-250 of SEQ ID NO: 6,wherein one or more of the amino acid residues at positions 107, 112,117, 159, 172, 173, 192, 197, 215, 256, 267, and 292 of SEQ ID NO: 6(Leu107, Lys112, Gln117, Lys159, Glu172, Glu173, Glu192, Asp197, Asp215,Lys256, Asp267, and Glu292) are mutated and/or modified. In any of thepreceding embodiments, the mutation(s) can substantially reduce oreliminate binding of the polypeptide to a NMI receptor or an inhibitoryantibody specific for NMI.

In some embodiments, disclosed herein is an isolated polypeptidecomprising, consisting essentially of, or consisting of the sequence setforth in amino acids 103-192, 201-292, or 151-240 of SEQ ID NO: 8,wherein one or more of the amino acid residues at positions 106, 111,116, 158, 171, 172, 191, 196, 214, 255, 266, and 291 of SEQ ID NO: 8(Leu106, Lys111, Gln116, Lys158, Glu171, Asp172, Asp191, Asp196, Asp214,Arg255, Asp266, and Asp291) are mutated and/or modified. In any of thepreceding embodiments, the mutation(s) can substantially reduce oreliminate binding of the polypeptide to a NMI receptor or an inhibitoryantibody specific for NMI.

In one aspect, disclosed herein is a pharmaceutical compositioncomprising the polypeptide of any one of the preceding embodiments. Insome embodiments, the pharmaceutical composition comprises apharmaceutically acceptable carrier or excipient.

In one aspect, disclosed herein is a method for stimulating an immuneresponse in a subject, and the method comprises administering, to asubject in need of such stimulation, an effective amount of thepolypeptide or the pharmaceutical composition of any one of thepreceding embodiments. In one aspect, the subject has a proliferationdisorder, a neoplasm, a tumor or a cancer. In another aspect, theproliferation disorder is selected from the group consisting of sarcoma,epidermoid cancer, fibrosarcoma, cervical cancer, gastric carcinoma,skin cancer, leukemia, lymphoma, lung cancer, non-small cell lungcancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renalcancer, prostate cancer, breast cancer, liver cancer, head and neckcancers, pancreatic cancer, bladder cancer and esophageal cancer. In anyof the preceding embodiments, the stimulation of the immune response cancomprise stimulation of the expression and/or activity of aninflammatory factor, the recruitment of immune cells such asneutrophils, and/or NF-κB signaling. In some aspects, the inflammatoryfactor comprises IL-1β, TNF-α, iNOS, and/or CD86, and the NF-κBsignaling is mediate by a TLR such as TLR4. In any one of the precedingembodiments, the stimulation of NF-κB signaling can comprise an increasein phosphorylated IκBα and/or a reduction in total IκBα.

In some aspects, provided herein are compositions and methods foractivating, boosting, augmenting, and/or potentiating immune response ina subject. The present compositions and methods can be used to treatand/or prevent any suitable disease or disorder associated withabnormally low level of a IFP35 family protein such as IFP35 and/or NMIin a subject. For example, the disease or disorder associated withabnormally low level of IFP35 and/or NMI in a subject can be aproliferation disorder, a neoplasm, a tumor or a cancer. Exemplaryproliferation disorders include sarcoma, epidermoid cancer,fibrosarcoma, cervical cancer, gastric carcinoma, skin cancer, leukemia,lymphoma, lung cancer, e.g., non-small cell lung cancer (NSCLC), coloncancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostatecancer, breast cancer, liver cancer, head and neck cancers, pancreaticcancer, bladder cancer and esophageal cancer.

In another aspect, disclosed herein is an isolated polynucleotideencoding a polypeptide comprising the polypeptide of any one of thepreceding embodiments. In yet another aspect, disclosed herein is anisolated vector comprising the polynucleotide of any one of thepreceding embodiments. In still another aspect, disclosed herein is anisolated host cell comprising the vector of any one of the precedingembodiments. In some aspects, the host cell is a eukaryotic cell such asa CHO cell. In some aspects, disclosed herein is a method of making anisolated polynucleotide, and the method comprises: a) culturing the hostcell under conditions suitable for expression of the polynucleotideencoding a polypeptide comprising the polypeptide of any of thepreceding embodiments; and b) isolating the polypeptide.

In some aspects, disclosed herein is a method of diagnosis, prognosis ortreatment monitoring of a disease or disorder associated with abnormallyhigh level and/or activity of IFP35 and/or NMI in a subject. In someaspects, the method comprises assessing the level and/or an activity ofIFP35 and/or NMI in a subject suspected of or being treated for adisease or disorder associated with abnormally high level and/oractivity of IFP35 and/or NMI. In one embodiment, the disease or disorderassociated with abnormally high level and/or activity of IFP35 and/orNMI is associated with excessive immune response. In another embodiment,the disease or disorder associated with abnormally high level and/oractivity of IFP35 and/or NMI is associated with cytokine storm. In anyof the preceding embodiments, the disease or disorder associated withabnormally high level and/or activity of IFP35 and/or NMI can beselected from the group consisting of inflammation, infection, organdamage, sepsis, and autoimmune disease.

In any of the preceding embodiments, the method can be used fordiagnosis of a disease or disorder associated with abnormally high leveland/or activity of IFP35 and/or NMI in a subject, and wherein a leveland/or an activity of IFP35 and/or NMI in a subject that is at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%,400%, or 500% higher than a level and/or an activity of IFP35 and/or NMIin a comparable subject that does not have a disease or disorderassociated with abnormally high level and/or activity of IFP35 and/orNMI, e.g., inflammation, infection, organ damage, sepsis, and autoimmunedisease, indicates that the subject has the disease or disorderassociated with abnormally high level and/or activity of IFP35 and/orNMI.

In any of the preceding embodiments, the method can further compriseassessing the level of IFP35 and/or NMI in a subject suspected of orbeing treated for a disease or disorder associated with abnormally highlevel and/or activity of IFP35 and/or NMI. In any of the precedingembodiments, the method can comprise assessing an activity of IFP35and/or NMI in a subject suspected of or being treated for a disease ordisorder associated with abnormally high level and/or activity of IFP35and/or NMI. In any of the preceding embodiments, the level and/or anactivity of IFP35 and/or NMI can be assessed at the DNA, RNA and/orprotein level. In one aspect, the level of IFP35 and/or NMI is assessedat the DNA and/or RNA level using a polynucleotide that is complementaryto at least 10 consecutive nucleotides in the IFP35 and/or NMI DNA orRNA. In another aspect, the level of IFP35 and/or NMI is assessed at theprotein level using an antibody that specifically binds to IFP35 and/orNMI.

Also disclosed herein, in one aspect, is a kit of diagnosis, prognosisor treatment monitoring of a disease or disorder associated withabnormally high level and/or activity of IFP35 and/or NMI in a subject.In some aspects, the kit comprises a means for assessing the leveland/or an activity of IFP35 and/or NMI in a subject suspected of orbeing treated for a disease or disorder associated with abnormally highlevel and/or activity of IFP35 and/or NMI.

Also disclosed herein, in one aspect, is a method of companiondiagnostics of a disease or disorder associated with abnormally highlevel and/or activity of IFP35 and/or NMI in a subject. In some aspects,the method comprises determining the genetic status of IFP35 and/or NMIgene in a subject being treated for the disease or disorder associatedwith abnormally high level and/or activity of IFP35 and/or NMI. In oneaspect, the genetic status of IFP35 and/or NMI gene in a subject isdetermined using a polynucleotide that is complementary to at least 10consecutive nucleotides in the IFP35 and/or NMI DNA or RNA.

Also disclosed herein, in one aspect, is a kit of companion diagnosticsof a disease or disorder associated with abnormally high level and/oractivity of IFP35 and/or NMI in a subject. In one aspect, the kitcomprises a means for determining the genetic status of IFP35 and/or NMIgene in a subject being treated for the disease or disorder associatedwith abnormally high level and/or activity of IFP35 and/or NMI.

In one aspect, disclosed herein is a method for identifying a modulatorof IFP35 and/or NMI. In some aspects, the method comprises: a)contacting IFP35 and/or NMI with a test substance and assessing anactivity of IFP35 and/or NMI that has been contacted by the testsubstance; b) assessing an activity of the IFP35 and/or NMI that has notbeen contacted by the test substance; and c) comparing the activities ofIFP35 and/or NMI assessed in steps a) and b), and identifying the testsubstance as a modulator of IFP35 and/or NMI when the activities ofIFP35 and/or NMI assessed in steps a) and b) are different. In oneaspect, the test substances are small molecules, a polypeptide librarycomprising mutants and/or fragments of IFP35 and/or NMI, antibodies thatspecifically bind IFP35 and/or NMI, a polynucleotide, such as an siRNA,shRNA, miRNA, or antisense RNAs. In any of the preceding embodiments,the method can be used to identify an inhibitor of an activity of IFP35and/or NMI. In any of the preceding embodiments, the method can be usedto identify a drug for treating and/or preventing a disease or disorderassociated with abnormal level and/or activity of IFP35 and/or NMI in asubject. In some aspects, the disease or disorder associated withabnormal level and/or activity of IFP35 and/or NMI is a proliferationdisorder, a neoplasm, a tumor or a cancer. In some embodiments, thedisease or disorder, such as a proliferation disorder, a neoplasm, atumor or a cancer, is associated with abnormally low level and/oractivity of a IFP35 family protein such as IFP35 or NMI.

In some aspects, also disclosed herein is a drug candidate identified bya method of any one of the preceding embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a crystal structure of NID-H (heterozygous NID). a.Truncations and putative domains of IFP35 are shown and the boundariesbetween domains are noted in the sequence. b. The structure of the IFP35dimer (closed conformational dimer, c-dimer) with secondary structureelements noted. c. Residues involved in the intra-molecule interactionsare labeled and shown in sticks. For clarity, only half of theinteractions are shown. d. The ring structure of the octamer, in whichmonomers are shown. e. Dimer with domain-swapping conformation (openconformational dimer, o-dimer) is highlighted with secondary structureelements noted on the upper panel. A NID-H molecule from c-dimer issuperimposed on the o-dimer as shown in the lower panel.

FIG. 2 shows the two stable aggregation states, dimer and octamer, ofNID-H. a. Size-exclusion chromatography analysis of NID-H usingSuperdex-200 (16/60 or 10/30) column (GE). The UV absorptions at 280 nmare shown in blue. The elution volumes of two peaks are 75 ml and 90 ml,suggesting dimer and octamer formation. b. O-dimer and c-dimersuperimposed on the octamer. Four o-dimers interact with one anotherthrough α1-α1 interactions, and form a ring like octamer structure. c.Structure comparison between NID-H and RNA recognition motif (RRM). TheR.M. S.D. between them is about 1.3 Å.

FIG. 3 shows that the open conformational dimer (o-dimer) of NID-H isresponsible for immune response stimulation. a. Octameric NID-Hup-regulates the transcription of TNF-α and IL-1β in RAW 264.7 cells.The cellular mRNA of TNF-α and IL-1β were detected by RT-PCR using GAPDHas internal control. b. Dimeric stated mutation on α1,K156E/K163E/R165E, partially up-regulates the transcription of TNF-α andIL-1β. c. IFP35 without the L-zip domain (ΔN) stimulated immune responsemore effectively than NID-H octamer. d. Similar with LPS, ΔN couldeffectively stimulate the TNF-α release of RAW264.7 cells. TNF-α levelsin cell culture were determined by ELISA after 4 h of incubation.

FIG. 4 shows that human source NMI protein up-regulates thetranscription of TNF-α and IL-1β in THP1 cells. PBS buffer was used asnegative control (NC). Results came from an average of threeindependently replicates. Error bars indicate the standard deviationamong them.

FIG. 5 shows IFP35 possesses characteristics of endogenous DAMPs. a.IFP35 released by RAW264.7 cells stimulated by LPS. RAW 264.7macrophages were pretreated with 100 ng/mL LPS. Cell lysate andsupernatants at 0, 1, 2, 3, 5, 9 hours were analyzed by western blot ofIFP35, HMGB1 and β-Actin. b. IFP35 is accumulated in the serum of LPSinduced septic mice. Serum concentrations of IFP35 in mice weredetermined by ELISA 3 h and 6 h after injection of LPS. c. IFP35 isaccumulated in the serum of septic patients. This data was obtained fromthe serum of 12 patients and 8 normal individuals. d. Survival rate ofthe septic mice administrated with IgG and IFP35 monoclonal antibodies(Anti-NID-H) was presented. Mice received LPS injection (i.p.) followedby treatment with anti-IFP35 antibody or IgG. Animal survival rate wasassessed in n=15 mice/group. e. Anti-NID-H neutralization attenuated therelease of early inflammatory cytokines such as IL-6, TNF-α and IL-1β,detected using ELISA. f. Survival of wild-type (n=12) and IFP35^(−/−)mice (n=12) treated with LPS and D-gal. g. The release of earlyinflammatory cytokines in the serum of IFP35 knock-out mice is reduced.Serum concentrations of IL-6, TNF-α and IL-1β in IFP35^(−/−) andwild-type mice as determined by ELISA 1 h and 3 h after injection of LPSand D-gal.

FIG. 6 shows IFP35 family members are released as endogenous DAMPs. a.IFP35 released by RAW cells stimulated by 10 ng/ml INF-γ. b. NMIreleased by RAW cells stimulated by Salmonella (SR). c and d. Theaggregation state of IFP35 (panel c) and NMI (panel d) were detected.After stimulating the RAW cells with SR, the cell culture were collectedand the released IFP35 family members were detected the by gelfiltration (Superdex-200 (16/60 or 10/30) column, GE) and western blot.The elution volume of IFP 35 is about 90 ml, as shown in panel c,suggesting a 35 kD monomer. NMI showed multiple aggregation statesdetected by gel filtration. The elution volume of its major conformationis about 76-82 ml, indicating that released NMI existed as dimer insolution. e. NMI is accumulated in the serum of LPS induced septic mice,as determined by ELISA 3 h and 6 h after injection of LPS. f. NMI isaccumulated in the serum of septic patients. Serum was obtained from 8healthy people and 12 septic patients. Serum concentrations of NMI weredetermined by ELISA. g. NMI knock-out mice is resistant to LPSchallenge. The survival rate of wide type (WT) (n=14) and Nmi^(−/−) mice(KO) (n=14) administrated with LPS was presented. h. The release ofinflammatory cytokines such as IL-6, TNF-α and IL-1β in the serum ofNmi^(−/−) mice is reduced. The results got from WT and KO mice are shownin gray and black, respectively. All the results were confirmed bymultiple independently analyzed biological replicates.

FIG. 7 shows IFP35 stimulates the cytokines storm based on TLR4/MD2/CD14complex. a. Octameric NID-H induced NF-κB promoter activity in a HEK293cell line transfected with TLR4, CD14, MD2 and NF-κB promoter luciferaseplasmid. 24 hours after transfection, cells were stimulated with 10μg/ml Octameric NID-H or 100 ng/ml LPS for 4 h (in the presence orabsence of 25 mg/ml polymyxin B) and assayed for luciferase activity.The luciferase activity trigged by LPS can be blocked by polymyxin B, aneffective antibiotic for Gram-negative infections. b. Purified IFP35(ΔN), a N-terminal truncation of IFP35 and LPS induce NF-κB and AP-1 butnot IRF3 promoter activity. The genes of TLR4, MD2 and CD14 and NF-κBpromoter (or AP-1, IRF3) with luciferase activity were transientlytransfected into HEK293 cells. IFP35 was used at 1 μg/ml, while 100ng/ml LPS were administrated as positive control.

FIG. 8 shows the structure model of tandem NID domains in IFP35. a andb. Secondary structure elements and sequence alignment of A and A′ aswell as B and B′ are shown in panel a. The identical residues arelabeled with “*”, while residues with similar features are labeled with“:” or “.”. Residues in the hydrophobic core of NID-H structure areshown in panel b which is highlighted in squares in panel a. c. Thedouble-barrel structure of tandem NIDs. Part B′ is shown in green andPart A′ is shown in cyan. The NID-H structure is shown in purple.

FIG. 9 shows sequence alignment of IFP35 and NMI generated by ClustalW.The identical residues are labeled with “*”, while residues with similarfeatures are labeled with “:” or “.”. The secondary structure elementsof NMI are predicted by PsiPred. The α-helixes and β-strands are shownin red and yellow, respectively. The two NID domains parallel to thosein IFP35 are labeled and highlighted in squares.

FIG. 10 illustrates the process of IFP35 protein purification.

FIG. 11 shows the different oligomer states of IFP35.

FIG. 12 shows the IFP35-NID crystal.

FIG. 13 shows the structure of the dimeric IFP35-NID.

FIG. 14 shows the structure of the octameric IFP35-NID.

FIG. 15 shows the structure of dimer with domain-swapping conformation.

FIG. 16 shows the residues of some areas.

FIG. 17 shows the purification of the recombinant NMI.

FIG. 18 reveals the expression level of IFP35 when stimulated bysalmonella.

FIG. 19 shows that the abundance of IFP35 changed with time as measuredby immunofluorescence.

FIG. 20 shows the abundance of IFP35 secreted to the medium at differentperiods when cells are infected by the virus.

FIG. 21 shows a model of mouse peritonitis.

FIG. 22 shows the contribution of the IFP35 with different oligomerstates during the process of stimulating inflammation.

FIG. 23 shows the result of Flow cytometry analysis, which illustratesoctameric IFP35-nid can recruit large amount of neutrophil granulocytes.

FIG. 24 shows exogenous octameric IFP35-NID can stimulate NF-κB pathwayin macrophages.

FIG. 25 illustrates that IFP35 can stimulate inflammation through myd88signal pathway.

FIG. 26 shows the levels of IL-1β and TNF among WT, TLR9−/− and TLR4−/−mouse as induced by IFP35.

FIG. 27A shows the amount of IFP35 in the serum of septic mice. FIG. 27Bshows the survival rate of the septic mice was increased whenadministrated with IFP35 monoclonal antibodies.

FIG. 28 shows that the block of IFP35 attenuated the release of inducedIL-6 by LPS in mice.

FIG. 29 shows the abundance of NMI in cell lysate and supernatant ofThp1 cells stimulated by salmonella.

FIG. 30 demonstrates that mouse source NMI protein can up-regulate thetranscription of TNFα and IL-1β in Thp1 cells.

FIG. 31 shows the detection of the aggregation state of NMI.

FIG. 32 shows a secondary structure prediction of human IFP35.

FIG. 33 shows the crystal structure coordinates of the NID-H dimer.

FIG. 34 shows the crystal structure coordinates of the NID-H octamer.

DETAILED DESCRIPTION OF THE INVENTION A. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,immunology, and pharmacology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,Molecular Cloning: A Laboratory Manual, 2^(nd) ed. (Sambrook et al.,1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal CellCulture (R. I. Freshney, ed., 1987); Methods in Enzymology (AcademicPress, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel etal., eds., 1987, and periodic updates); PCR: The Polymerase ChainReaction (Mullis et al., eds., 1994); and Remington, The Science andPractice of Pharmacy, 20^(th) ed., (Lippincott, Williams & Wilkins2003).

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, patentapplications (published or unpublished), and other publications referredto herein are incorporated by reference in their entireties. If adefinition set forth in this section is contrary to or otherwiseinconsistent with a definition set forth in the patents, applications,published applications and other publications that are hereinincorporated by reference, the definition set forth in this sectionprevails over the definition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength, e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300,400, 500, 1,000 or more amino acids. The polymer may be linear orbranched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The terms also encompass an amino acidpolymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art.

As used herein, the terms “variant” is used in reference to polypeptidesthat have some degree of amino acid sequence identity to a parentpolypeptide sequence. A variant is similar to a parent sequence, but hasat least one substitution, deletion or insertion in their amino acidsequence that makes them different in sequence from a parentpolypeptide. Additionally, a variant may retain the functionalcharacteristics of the parent polypeptide, e.g., maintaining abiological activity that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%,or 99% of that of the parent polypeptide.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule, and can be animmunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE. IgY,which is the major antibody type in avian species such as chicken, isalso included within the definition. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain(ScFv), mutants thereof, naturally occurring variants, fusion proteinscomprising an antibody portion with an antigen recognition site of therequired specificity, humanized antibodies, chimeric antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity.

As used herein, the term “antigen” refers to a target molecule that isspecifically bound by an antibody through its antigen recognition site.The antigen may be monovalent or polyvalent, i.e., it may have one ormore epitopes recognized by one or more antibodies. Examples of kinds ofantigens that can be recognized by antibodies include polypeptides,oligosaccharides, glycoproteins, polynucleotides, lipids, etc.

As used herein, the term “epitope” refers to a portion of an antigen,e.g., a peptide sequence of at least about 3 to 5, preferably about 5 to10 or 15, and not more than about 1,000 amino acids (or any integerthere between), which define a sequence that by itself or as part of alarger sequence, binds to an antibody generated in response to suchsequence. There is no critical upper limit to the length of thefragment, which may, for example, comprise nearly the full-length of theantigen sequence, or even a fusion protein comprising two or moreepitopes from the target antigen. An epitope for use in the subjectinvention is not limited to a peptide having the exact sequence of theportion of the parent protein from which it is derived, but alsoencompasses sequences identical to the native sequence, as well asmodifications to the native sequence, such as deletions, additions andsubstitutions (conservative in nature).

As used herein, the term “specifically binds” refers to the bindingspecificity of a specific binding pair. Recognition by an antibody of aparticular target in the presence of other potential targets is onecharacteristic of such binding. Specific binding involves two differentmolecules wherein one of the molecules specifically binds with thesecond molecule through chemical or physical means. The two moleculesare related in the sense that their binding with each other is such thatthey are capable of distinguishing their binding partner from otherassay constituents having similar characteristics. The members of thebinding component pair are referred to as ligand and receptor(anti-ligand), specific binding pair (SBP) member and SBP partner, andthe like. A molecule may also be an SBP member for an aggregation ofmolecules; for example an antibody raised against an immune complex of asecond antibody and its corresponding antigen may be considered to be anSBP member for the immune complex.

As used herein, a “tag” or an “epitope tag” refers to a sequence ofamino acids, typically added to the N- and/or C-terminus of apolypeptide. The inclusion of tags fused to a polypeptide can facilitatepolypeptide purification and/or detection. Typically a tag or tagpolypeptide refers to polypeptide that has enough residues to provide anepitope recognized by an antibody or can serve for detection orpurification, yet is short enough such that it does not interfere withactivity of chimeric polypeptide to which it is linked. The tagpolypeptide typically is sufficiently unique so an antibody thatspecifically binds thereto does not substantially cross-react withepitopes in the polypeptide to which it is linked. Suitable tagpolypeptides generally have at least 5 or 6 amino acid residues andusually between about 8-50 amino acid residues, typically between 9-30residues. The tags can be linked to one or more chimeric polypeptides ina multimer and permit detection of the multimer or its recovery from asample or mixture. Such tags are well known and can be readilysynthesized and designed. Exemplary tag polypeptides include those usedfor affinity purification and include His tags, the influenzahemagglutinin (HA) tag polypeptide and its antibody 12CA5; the c-myc tagand the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto; and theHerpes Simplex virus glycoprotein D (gD) tag and its antibody. See,e.g., Field et al. (1988) Mol. Cell. Biol. 8:2159-2165; Evan et al.(1985) Mol. Cell. Biol. 5:3610-3616; Paborsky et al. (1990) ProteinEngineering 3:547-553.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidemay comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure maybe imparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupsmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example,2′-O-methyl-2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugaranalogs, α-anomeric sugars, epimeric sugars such as arabinose, xylosesor lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), “(O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

As used herein, the term “homologue” is used to refer to a nucleic acidwhich differs from a naturally occurring nucleic acid (e.g., the“prototype” or “wild-type” nucleic acid) by minor modifications to thenaturally occurring nucleic acid, but which maintains the basicnucleotide structure of the naturally occurring form. Such changesinclude, but are not limited to: changes in one or a few nucleotides,including deletions (e.g., a truncated version of the nucleic acid)insertions and/or substitutions. A homologue can have enhanced,decreased, or substantially similar properties as compared to thenaturally occurring nucleic acid. A homologue can be complementary ormatched to the naturally occurring nucleic acid. Homologues can beproduced using techniques known in the art for the production of nucleicacids including, but not limited to, recombinant DNA techniques,chemical synthesis, etc.

As used herein, “substantially complementary or substantially matched”means that two nucleic acid sequences have at least 90% sequenceidentity. Preferably, the two nucleic acid sequences have at least 95%,96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively,“substantially complementary or substantially matched” means that twonucleic acid sequences can hybridize under high stringency condition(s).

In general, the stability of a hybrid is a function of the ionconcentration and temperature. Typically, a hybridization reaction isperformed under conditions of lower stringency, followed by washes ofvarying, but higher, stringency. Moderately stringent hybridizationrefers to conditions that permit a nucleic acid molecule such as a probeto bind a complementary nucleic acid molecule. The hybridized nucleicacid molecules generally have at least 60% identity, including forexample at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity.Moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. Highstringency conditions can be provided, for example, by hybridization in50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C.,followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. Low stringencyhybridization refers to conditions equivalent to hybridization in 10%formamide, 5×Denhardt's solution, 6×SSPE, 0.2% SDS at 22° C., followedby washing in 1×SSPE, 0.2% SDS, at 37° C. Denhardt's solution contains1% Ficoll, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA).20×SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraaceticacid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and0.025 M (EDTA). Other suitable moderate stringency and high stringencyhybridization buffers and conditions are well known to those of skill inthe art.

As used herein, the term “RNA interference” or “RNAi” refers generallyto a process in which a double-stranded RNA molecule or a short hairpinRNA molecule reducing or inhibiting the expression of a nucleic acidsequence with which the double-stranded or short hairpin RNA moleculeshares substantial or total homology. The term “short interfering RNA”or “siRNA” or “RNAi agent” refers to an RNA (or RNA analog) sequencecomprising between about 10-50 nucleotides (or nucleotide analogs) thatelicits RNA interference. See Kreutzer et al., WO 00/44895;Zernicka-Goetz et al., WO 01/36646; Fire, WO 99/32619; Mello & Fire, WO01/29058. As used herein, siRNA molecules include RNA moleculesencompassing chemically modified nucleotides and non-nucleotides. Theterm “ddRNAi agent” refers to a DNA-directed RNAi agent that istranscribed from an exogenous vector. The terms “short hairpin RNA” or“shRNA” refer to an RNA structure having a duplex region and a loopregion. In certain embodiments, ddRNAi agents are expressed initially asshRNAs.

As used herein, “vector (or plasmid)” refers to discrete elements thatare used to introduce heterologous DNA into cells for either expressionor replication thereof. Selection and use of such vehicles are wellknown within the skill of the artisan. An expression vector includesvectors capable of expressing DNA's that are operatively linked withregulatory sequences, such as promoter regions, that are capable ofeffecting expression of such DNA fragments. Thus, an expression vectorrefers to a recombinant DNA or RNA construct, such as a plasmid, aphage, recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, “a promoter region or promoter element” refers to asegment of DNA or RNA that controls transcription of the DNA or RNA towhich it is operatively linked. The promoter region includes specificsequences that are sufficient for RNA polymerase recognition, bindingand transcription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences that modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be cis actingor may be responsive to trans acting factors. Promoters, depending uponthe nature of the regulation, may be constitutive or regulated.Exemplary promoters contemplated for use in prokaryotes include thebacteriophage T7 and T3 promoters, and the like.

As used herein, “operatively linked or operationally associated” refersto the functional relationship of DNA with regulatory and effectorsequences of nucleotides, such as promoters, enhancers, transcriptionaland translational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itmay be necessary to remove, add or alter 5′ untranslated portions of theclones to eliminate extra, potential inappropriate alternativetranslation initiation (i.e., start) codons or other sequences that mayinterfere with or reduce expression, either at the level oftranscription or translation. Alternatively, consensus sites can beinserted immediately 5′ of the start codon and may enhance expression.See, e.g., Kozak (1991) J. Biol. Chem. 266:19867-19870. The desirabilityof (or need for) such modification may be empirically determined.

“Treating” or “treatment” or “alleviation” refers to therapeutictreatment wherein the object is to slow down (lessen) if not cure thetargeted pathologic condition or disorder or prevent recurrence of thecondition. A subject is successfully “treated” if, after receiving atherapeutic amount of a therapeutic agent or treatment, the subjectshows observable and/or measurable reduction in or absence of one ormore signs and symptoms of the particular disease. Reduction of thesigns or symptoms of a disease may also be felt by the patient. Apatient is also considered treated if the patient experiences stabledisease. In some embodiments, treatment with a therapeutic agent iseffective to result in the patients being disease-free 3 months aftertreatment, preferably 6 months, more preferably one year, even morepreferably 2 or more years post treatment. These parameters forassessing successful treatment and improvement in the disease arereadily measurable by routine procedures familiar to a physician ofappropriate skill in the art. In some embodiments, “treatment” means anymanner in which the symptoms of a condition, disorder or disease areameliorated or otherwise beneficially altered. Treatment alsoencompasses any pharmaceutical use of the compositions herein. In someembodiments, “amelioration” of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient that canbe attributed to or associated with administration of the composition.

The term “prediction” or “prognosis” is used herein to refer to thelikelihood that a patient will respond either favorably or unfavorablyto a drug or set of drugs, or the likely outcome of a disease. In oneembodiment, the prediction relates to the extent of those responses oroutcomes. In one embodiment, the prediction relates to whether and/orthe probability that a patient will survive or improve followingtreatment, for example treatment with a particular therapeutic agent,and for a certain period of time without disease recurrence. Thepredictive methods of the invention can be used clinically to maketreatment decisions by choosing the most appropriate treatmentmodalities for any particular patient. The predictive methods of thepresent invention are valuable tools in predicting if a patient islikely to respond favorably to a treatment regimen, such as a giventherapeutic regimen, including for example, administration of a giventherapeutic agent or combination, surgical intervention, steroidtreatment, etc.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. See, e.g.,Remington, The Science and Practice of Pharmacy, 20^(th) ed.,(Lippincott, Williams & Wilkins 2003). Except insofar as anyconventional media or agent is incompatible with the active compound,such use in the compositions is contemplated.

A “pharmaceutically acceptable salt” is intended to mean a salt of afree acid or base of a compound represented herein that is non-toxic,biologically tolerable, or otherwise biologically suitable foradministration to the subject. See, generally, Berge, et al., J. Pharm.Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts arethose that are pharmacologically effective and suitable for contact withthe tissues of subjects without undue toxicity, irritation, or allergicresponse. A compound described herein may possess a sufficiently acidicgroup, a sufficiently basic group, both types of functional groups, ormore than one of each type, and accordingly react with a number ofinorganic or organic bases, and inorganic and organic acids, to form apharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts include sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrogen-phosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,methylsulfonates, propylsulfonates, besylates, xylenesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates,phenylpropionates, phenylbutyrates, citrates, lactates,γ-hydroxybutyrates, glycolates, tartrates, and mandelates.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of a therapeutic agent that whenadministered alone or in combination with an additional therapeuticagent to a cell, tissue, or subject is effective to prevent orameliorate a disease or disorder associated with abnormally high levelof IFP35 and/or NMI in a subject. A therapeutically effective dosefurther refers to that amount of the therapeutic agent sufficient toresult in amelioration of symptoms, e.g., treatment, healing, preventionor amelioration of the relevant medical condition, or an increase inrate of treatment, healing, prevention or amelioration of suchconditions. When applied to an individual active ingredient administeredalone, a therapeutically effective dose refers to that ingredient alone.When applied to a combination, a therapeutically effective dose refersto combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. In some embodiment, “an effective amount of a compoundfor treating a particular disease” is an amount that is sufficient toameliorate, or in some manner reduce the symptoms associated with thedisease. Such amount may be administered as a single dosage or may beadministered according to a regimen, whereby it is effective. The amountmay cure the disease but, typically, is administered in order toameliorate the symptoms of the disease. Repeated administration may berequired to achieve the desired amelioration of symptoms.

The term “combination” refers to either a fixed combination in onedosage unit form, or a kit of parts for the combined administrationwhere a compound and a combination partner (e.g., another drug asexplained below, also referred to as “therapeutic agent” or “co-agent”)may be administered independently at the same time or separately withintime intervals, especially where these time intervals allow that thecombination partners show a cooperative, e.g., synergistic effect. Theterms “co-administration” or “combined administration” or the like asutilized herein are meant to encompass administration of the selectedcombination partner to a single subject in need thereof (e.g., apatient), and are intended to include treatment regimens in which theagents are not necessarily administered by the same route ofadministration or at the same time. The term “pharmaceuticalcombination” as used herein means a product that results from the mixingor combining of more than one active ingredient and includes both fixedand non-fixed combinations of the active ingredients. The term “fixedcombination” means that the active ingredients, e.g., a compound and acombination partner, are both administered to a patient simultaneouslyin the form of a single entity or dosage. The term “non-fixedcombination” means that the active ingredients, e.g., a compound and acombination partner, are both administered to a patient as separateentities either simultaneously, concurrently or sequentially with nospecific time limits, wherein such administration providestherapeutically effective levels of the two compounds in the body of thepatient. The latter also applies to cocktail therapy, e.g., theadministration of three or more active ingredients.

As used herein, “biological sample” refers to any sample obtained from aliving or viral source or other source of macromolecules andbiomolecules, and includes any cell type or tissue of a subject fromwhich nucleic acid or protein or other macromolecule can be obtained.The biological sample can be a sample obtained directly from abiological source or a sample that is processed. For example, isolatednucleic acids that are amplified constitute a biological sample.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples from animals and plants and processedsamples derived therefrom.

The terms “level” or “levels” are used to refer to the presence and/oramount of a target, e.g., a protein or polynucleotide, and can bedetermined qualitatively or quantitatively. A “qualitative” change inthe target, protein or polynucleotide level refers to the appearance ordisappearance of a target, protein or polynucleotide that is notdetectable or is present in samples obtained from normal controls. A“quantitative” change in the levels of one or more targets, proteins orpolynucleotides refers to a measurable increase or decrease in thetarget, protein or polynucleotide levels when compared to a healthycontrol.

A “healthy control” or “normal control” is a biological sample takenfrom an individual who does not suffer from a disease or disorderassociated with abnormally high level of IFP35 and/or NMI. A “negativecontrol” is a sample that lacks any of the specific analyte the assay isdesigned to detect and thus provides a reference baseline for the assay.

As used herein, “mammal” refers to any of the mammalian class ofspecies. Frequently, the term “mammal,” as used herein, refers tohumans, human subjects or human patients. “Mammal” also refers to any ofthe non-human mammalian class of species, e.g., experimental, companionor economic non-human mammals. Exemplary non-human mammals include mice,rats, rabbits, cats, dogs, pigs, cattle, sheep, goats, horses, monkeys,Gorillas and chimpanzees.

As used herein, “production by recombinant means” refers to productionmethods that use recombinant nucleic acid methods that rely onwell-known methods of molecular biology for expressing proteins encodedby cloned nucleic acids.

As used herein, the term “subject” is not limited to a specific speciesor sample type. For example, the term “subject” may refer to a patient,and frequently a human patient. However, this term is not limited tohumans and thus encompasses a variety of non-human animal or mammalianspecies.

As used herein, a “prodrug” is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug may be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388-392).

As used herein, “test substance (or candidate compound)” refers to achemically defined compound (e.g., organic molecules, inorganicmolecules, organic/inorganic molecules, proteins, peptides, nucleicacids, oligonucleotides, lipids, polysaccharides, saccharides, orhybrids among these molecules such as glycoproteins, etc.) or mixturesof compounds (e.g., a library of test compounds, natural extracts orculture supernatants, etc.) whose effect on IFP35 and/or NMI isdetermined by the disclosed and/or claimed methods herein.

As used herein, high-throughput screening (HTS) refers to processes thattest a large number of samples, such as samples of diverse chemicalstructures against disease targets to identify “hits” (see, e.g.,Broach, et al., High throughput screening for drug discovery, Nature,384:14-16 (1996); Janzen, et al., High throughput screening as adiscovery tool in the pharmaceutical industry, Lab Robotics Automation:8261-265 (1996); Fernandes, P. B., Letter from the society president, J.Biomol. Screening, 2:1 (1997); Burbaum, et al., New technologies forhigh-throughput screening, Curr. Opin. Chem. Biol., 1:72-78 (1997)). HTSoperations are highly automated and computerized to handle samplepreparation, assay procedures and the subsequent processing of largevolumes of data.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Throughout this disclosure, various aspects of this invention arepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Other objects, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

C, Methods for Treating and/or Preventing a Disease or DisorderAssociated with Abnormally High Level and/or Activity of the IFP35Family of Proteins

The IFP35 family proteins, including IFP35 and NMI, can be rapidlyup-regulated by type I or type II interferon in numerous immune cells.Yang et al., PLoS One 7, e50932 (2012); Das et al., Journal of Virology88, 3103-3113 (2014); Zhu et al., Cell 96, 121-130 (1999); and Lebrun etal., Journal of Interferon & Cytokine Research 18, 767-771 (1998). Thefunctions of IFP35 family members are not clear. IFP35 family membersare predicted to consist of a Leucine-zipper (L-zip) domain in theirN-termini, followed by two tandem structural and functional unknownNMI/IFP35 domains (NIDs). In the early report, people found IFP35 andNMI located in the cytoplasm using immunofluorescence microscopy. Lebrunet al., Journal of Interferon & Cytokine Research 18, 767-771 (1998).They could assemble into high molecular mass complex (HMMC) mediated bytheir NID domains and further into functional unknown speckle-likeaggregations. Zhou et al., JBC 275, 21364-21371 (2000); and Chen et al.,JBC 275, 36278-84 (2000). It was known that IFP35 could recognizespecific viral nucleic acids from bovine foamy virus, and inhibit thevirus transcription. Tan et al., Journal of Virology 82, 4275-4283(2008). However, recent results suggested that it could supportvesicular stomatitis virus replication, by specifically interacted withRIG-1 (retinoic acid-inducible gene I) and negatively regulated the hostinnate immune response. Das et al., Journal of Virology 88, 3103-3113(2014). Investigations of NMI mainly focused on its inhibitory effectson the proliferation and metastasis of cancer cells, by regulatingmultiple signaling pathways. Li et al., Mol Biol Cell 23, 4635-4646(2012); Fillmore et al., International Journal of Cancer 125, 556-64(2009); and Li et al., JBC 277, 20965-73 (2002). To further explore thepotential functions and mechanisms of IFP35 family members, theinventors determined the X-ray crystallography structure of the NIDdomain in IFP35. It was found that IFP35 and NMI could serve as DAMPsfor immune modulation. Some DAMP proteins were reported to be recognizedby specific cell-surface receptor, TLR4, with the help of MD2 and otherassociated proteins. See Yang et al., PNAS 107, 11942-11947 (2010);Nagai et al., Nature Immunology 3, 667-672 (2002); and Zanoni et al.,Cell 147, 868-880 (2011). A complex signal cascade can be trigged toactivate the transcription factor NF-κB, resulting in cytokines releaseand enhanced host inflammatory response. See Meylan et al., NatureImmunology 5, 503-507 (2004); Moynagh, Trends in Immunology 26, 469-476(2005); and Lu et al., Cytokine 42, 145-151 (2008).

In some aspects, disclosed herein is a use of a truncated IFP35 protein(e.g., octamer of the truncated protein) or a truncated NMI protein inpreparation of an agent for regulating the inflammatory response. Insome aspects, disclosed herein is a use of a truncated IFP35 protein(e.g., octamer of the truncated protein) or a truncated NMI protein inpreparation of antineoplastic products. In some aspects, by using aIFP35 antibody or NMI antibody to neutralize IFP35 or NMI in cells orthe humoral fluids, it can effectively regulate IFP35 or NMI proteinoverexpression-induced excessive inflammatory response, inhibitinflammatory effect, avoid the generation of body and tissue damage byinflammation and dramatically improve the survival rate of a subject,such as a subject with a bacterial or viral infection or autoimmunedisease. In some embodiments, the subject is with a viral infection(such as influenza virus, SARS, MERS, HBV, HCV), bacterial infection(such as Mycobacterium tuberculosis), fungal infection, and/or infectioncaused by other pathogens. In some aspects, the infections provokeexpression of a IFP35 family protein, such as IFP35 and/or NMIexpression. Overexpression of IFP35 and/or NMI in the infected patientsmay lead to severe damage of their organs and bodies. Abnormally highlevel of IFP35 may lead to tissue or organ damage because of provokingof cytokine storm.

Thus, the present compositions and/or methods can be used to treat,ameliorate, and/or prevent a number of infectious diseases, infectionstates, inflammation, autoimmune disease, graft-versus-host disease, orconditions associated with over-activation of the immune system in asubject. Pathogenic viruses include, but are not limited to,Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III); and otherisolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitisA virus; enteroviruses, human coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g., strains that cause gastroenteritis);Togaviridae (e.g., equine encephalitis viruses, rubella viruses);Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g.,vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebolaviruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.,influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Bimaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses); Poxyiridae (variola viruses, vaccinia viruses, pox viruses);and Iridoviridae (e.g., African swine fever virus); Hepatitis C virus;and unclassified viruses (e.g., the agent of delta hepatitis (thought tobe a defective satellite of hepatitis B virus); Norwalk and relatedviruses, and astroviruses).

Pathogenic bacteria include, but are not limited to, Helicobacterpyloris, Borelia burgdorferi, Legionella pneumophila, Mycobacteria sps(e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M.gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseriameningitidis, Listeria monocytogenes, Streptococcus pyrogenes (Group AStreptococcus), Streptococcus agalactiae (Group B Streptococcus),Streptococcus (viridans group), Streptococcus faecalis, Streptococcusbovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae,pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae,Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurellamultocida, Bacteroides sp., Fusobacterium nucleatum, pathogenic strainsof Escherichia coli, Streptobacillus moniliformis, Treponema pallidium,Treponema pertenue, Leptospira, and Actinomyces israelli.

Infectious fungi include, but are not limited to, Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis, Chlamydia trachomatis, Candida albicans.

Infectious protozoa include, but are not limited to, Plasmodium spp.,e.g., Plasmodium falciparum; Trypanosomes, e.g., Trypanosoma cruzi; andToxoplasma gondii.

Allergens include, but are not limited to, pollens, insect venoms,animal dander dust, fungal spores and drugs (e.g. penicillin). Examplesof natural, animal and plant allergens include proteins specific to thefollowing genera: Canine (Canis familiaris); Dermatophagoides (e.g.Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosiaartemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum);Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata);Alder; Alnus (Alnus gultinosa); Betula (Betula verrucosa); Quercus(Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris);Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietariaofficinalis or Parietaria judaica); Blattella (e.g. Blattellagermanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressussempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus(e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis andJuniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.Charnaecyparis obtusa); Periplaneta (e.g. Periplaneta americana);Agropyron(e.g. Agropyron repens); Secale (e.g. Secale cereale); Triticum(e.g. Triticum aestivum); Dactylis (e.g. Dactylis glomerata);Festuca(e.g. Festuca elatior); Poa (e.g. Poa pratensis or Poacompressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus);Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g.Arrhenatherum elatius); Agrostis(e.g. Agrostis alba); Phleum (e.g.Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g.Paspalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g.Bromus inermis). Use of epitopes from the above allergens in the presentmethods for antibody detection and analysis is also envisaged.

In some aspects, the present compositions and/or methods can also beused for relieving disease-induced or associated suppression of asubject's immune system, such as cancer-induced or associatedsuppression of the subject's immune system. For example, byadministering a IFP35 family protein or analog or derivative therefore,the subject's immune response can be activated and/or augmented in orderto overcome the disease-causing vector's ability to evade an immunereaction. This therapeutic approach is applicable to disease stateswhereby immune system suppression has been demonstrated, such asbacterial infections, parasitic infections including malaria andtypanosomiasis, viral infections, peptic ulcers and gastric cancer dueto H. Pylori infection together with prevention of pregnancy.

In some aspects, provided herein are the amino acid residues that areinvolved in binding with a IFP35 receptor or an antagonist antibodyagainst IFP35. In one aspect, the domain-swapping dimer form an arcintersecting surface area, and the dimer provides some residues such asSer145, Asp172, Val1173, Leu177, Arg212, Gln199, Gln207, Gln208, Pro210,Ser214, Thr201, and Tyr216 to form a large exposed surface as shown inFIG. 16A. Among the residues, residues Glu150, Glu175, Leu177, Gln207,and Gln208 are in a more prominent position (FIG. 16A). Consequently,they are more likely to bind the receptor or antibody.

In some aspects, other residues on the inner surface of the ringstructure formed by the IFP5-NID octamer participate in the interactionwith other proteins. These residues can include Ser145, Arg147, Glu150,Glu151, Val173, Gly206, Gln207, and Gln208. In some aspects, Arg147,Gln207, Gln208, Glu150, and Glu151 provide extended side chains thatprotrude from the inner surface of octamer ring structure (FIG. 16B).

Based on the structure of the dimer or the octamer, there are somerelatively large amino acid residues near the top of the amino terminusand the carboxyl terminus of the 3 sheet barrel-like structure of thesingle monomer or domain-swapping monomer; these relatively large aminoacid residues extend outwards. These residues can include Arg187,Glu188, Gln192, Gln196, Arg212, and Tyr216, and they are close indistance. Furthermore, some amino acid residues may participate inbinding to other proteins, antibodies, or small molecules (FIG. 16C).

These residues above are located on the surface of the structure. Insome embodiments, these residues participate in binding with thereceptor or the antibody and are the key position for drug inhibition.

In some aspects, the present inventors have expressed, purified, andcrystallized IFP35 and NMI, and have also determined the structure ofone fragment of IFP35. The fragment of IFP35 can induce inflammatoryresponse and further IFP35 and NMI can be secreted out from the cell tostimulate intense inflammatory response. It can be effective to controlthe excessive inflammatory response caused by the excessive secretoryexpression of IFP35 or NMI through neutralization of IFP 35 or NMI incell solution or body fluids using a IFP35 antibody or a NMI antibody.In some aspects, the present disclosure avoids the damage of the bodyand the tissue and raises the survival rates of the subjects. Theresults indicate that a disease or condition associated with theexcessive expression of IFP35 and NMI, such as bacterial infections,virus invasions, autoimmune diseases, tumor, and other infections, maybe controlled by the reduction of the concentration of IFP35 and/or NMI.Meanwhile, diseases caused by the defect or deficiency of the expressionand/or procession (such as secretion) of IFP35 and/or NMI may beimproved through the addition of the proteins.

Below is a sequence alignment of human IFP35 (hIFP), mouse IFP35 (mIFP),human NMI (hNMI) and mouse NMI (mNMI), using CLUSTAL 2.1 multiplesequence alignment.

hNMI MEADKDDTQQILKEHSPD-EFIKDEQNKGLIDEITKKNIQLKKEIQKLETELQEATKEFQ  59mNMI MDADKDNIKQACDERSAEMDDMRGEQSMGLVHEIMSENKELDEEIKKLEAELQSDAREFQ  60hIFP ----------------------MSAPLDAALHALQEEQARLKMRLWDLQQLRKELGDSPK  38mIFP ----------------------MSVTLQTVLYSLQEEQARLKMRLQELQQLKRERTGSPG  38.      :  : .:: .*. .: .*:   :.   . hNMIIKEDIPETKMKELSVETPENDSQLSNISCSFQVSSKVPYEIQKGQALITFEKEEVAQNVV 119 mNMIIKENVPEKKLKLTSVESPKDGCHFSNSSCSFQVSSQILYELQEGQALITFEKEEVAQNVI 120 hIFPDKVPFSVPKIPLVERGHTQQDPEVPKSLVS---NLRIHOPLLAGSALITEDDPKVAEQVL  95 mIFPAKIPFSVPEVPLVFQGQTKQGRQVPKFVVS---NLKVCCPLPEGSALVTFEDPKVVDRLL  95*  ..  :: : .::. ...:   *   . ::   :  *.**:**:. :*.:.:: hNMISMSKHHVQMIDVNLEVTAKPVPLNSGVREQVY--VEVSKMKINVTEIPD--TLREDQMRD 175 mNMISMGNHVVQMEGTPVKVSAHPVPLNTGVREQVH--VDISKMKINVTGIPD--ELSEEQTRD 176 hIFPQQKEHTINMEECRLRVQVQPLELPMVTTIQVMMSSQLSGRRVLVTGFPASLRLSEEELLD 155 mIFPQQKEHRVNLEDCRLRVQVQPLELPVVTNIQV--SSQPDNHRVLVSGFPAGLRLSEEELLD 153.  :* ::::   :.*  .:*: *   . :**    : .  :: *: :*    * *::  * hNMIKLELSFSKSRNGGGEVDRVDYDRQSGSAVITFVEIGVADKILKKKEYPLYINQTCHRVTV 235 mNMIKLELSECKSRNGGGEVESVDYDRKSRSAVITFVETGVVDKILKKKTYPLYMNQKCHSVAV 236 hIFPKLEIFFGKTRNGGGDVDVR--ELLPGSVMLGFARDGVAQRLCQIGQFTVPLGGQQVPLRV 213 mIFPKLEIFFGKAKNGGGDVETR--EMLQGTVMLGFADEEVAQHLCQIGQFRVPLDRQQVLLRV 211***: * *::****:* :    :    :.:: *.   *.::: :   : : :.     : * hNMISPYTEIHLKKYQIFSGTSKRTVLLTGMEGIQMDEEIVEDLINIHFQRAKNGGGEVDVVKC 295 mNMISPCIERCLEKYQVFSAVSKKTVLLTGLEGIPVDEETGEDLLNIHFQRKNNGGGEVEVVKC 296 hIFPSPYVNGEIQKAEIRSQPVPRSVLVLNIPDI-LDGPELHDVLETHFQKPTRGGGEVEALTV 272 mIFPSPYVSGEIQKAEIKFQQAPHSVLVTNIPDV-MDAQELHDILEIHFQKPTRGGGEVEALTV 270**  .  ::* ::    ::**: .: .: :*    .*:::****: ..*****:.:. hNMIS-LGQPHIAYFEE------ 307 mNMI S-LDQSFAAYEKEEARETI 314 hIFPVPQGQQGLAVFTSESG--- 288 mIFP VPSGQQGLAIFTSESS--- 286 .*   * * .

In some embodiments, provided herein is a method of using a IFP35 familyprotein in provoking an immune response. In another embodiment, providedherein is a method of inhibiting and/or reducing a IFP35 family protein,such as IFP35 and NMI, in order to inhibit cytokine storm, such as byinfection of bacteria, viruses or organ damaging induced by abnormalhigh level of IFP35/NMI by food or drug. The organ may include lung,kidney, liver or others organs.

In some embodiments, provided herein is an antagonist compound for aIFP35 family protein, such as an antibody or a small molecule compound,for treating or preventing a disease or condition associated with orcaused by abnormal levels of the IFP35 family protein.

In other embodiments, provided herein is a method for applying thecrystal structure method for further designing and/or selecting anantibody or compound. In one aspect, the structure is used to see howdrugs or antibody binds to the target protein, and then the antibody orcompound can be further modified. From the target protein/antibodyco-crystal structure, epitope of the target protein can also beidentified.

Embodiment 1

Application of truncated IFP35 protein in preparing reagents/products toprovoke immune response and inflammation or in preparing anti-tumorproducts, wherein said truncated IFP35 is human IFP35 or mouse IFP35,wherein said truncated human IFP35 is as 1) or 2) below:

1) protein with residues as in SEQ ID NO: 2, from residues 136-216;

2) said protein sequence as in 1), wherein 1-10 additional residues areadded to the sequence's amino or/and carboxyl terminus, and the sequencehomology is at least about 90% to the sequence of 1);

wherein said truncated mouse IFP35 is as 3) or 4) below:

3) protein with residues as in SEQ ID NO: 4, from residues 134-216;

4) said protein sequence as in 3), wherein 1-10 additional residues areadded to the sequence's amino or/and carboxyl terminus, and the sequencehomology is at least about 90% to the sequence of 3).

Embodiment 2

A method of Embodiment 1, wherein the truncated human IFP35 protein asshown in SEQ ID NO: 2, from residues 124-220.

Embodiment 3

A method in preparing products to provoke immune response/inflammationby IFP35 protein or in preparing products to against tumor/cancer,wherein said sequence of IFP35 protein is shown as in SEQ ID NO: 2 orSEQ ID NO: 4.

Embodiment 4

A method in preparing products to provoke immune response/inflammationby IFP35 mutant proteins or in preparing products to againsttumor/cancer, wherein said IFP35 mutant protein has at least onemutation and/or modification of a residue involved in binding a IFP35receptor or an inhibitory antibody, which mutation and/or modificationleads to reduced or no binding of IFP35 to the IFP35 receptor orinhibitory antibody, wherein said residue involved in receptorbinding/antibody binding is Ser145, Asp172, Val 173, Leu 177, Arg212,Gln199, Gln207, Gin 208, Pro210, Ser214, Thr201, Tyr216, Glu150, Glu175,Gln208, Arg147, Glu151, or Gly206, or any combination thereof. Accordingto the structure comparison results, corresponding position residuesthat are in sequences from other sources of IFP35 and NMI are within thepresent disclosure. According to the structure comparison results, IFP35and NMI both have two NID domains, these two NID domains are homologs toeach other, therefore, corresponding NID region residues of IFP35 andNMI outside the structure determined IFP35 NID domain are within thepresent disclosure.

Embodiment 5

The method according to any of Embodiments 1-4, wherein the methodinduces immune response/inflammation to increase expression ofinflammation factors, recruitments of neutrophils or stimulate NF-κBpathway, wherein said inflammation factors include: IL-1β, TNF-α, iNOS,or CD86, and wherein said stimulating NF-κB pathway is to increase IκBαto reduce the total IκBα amount.

Embodiment 6

A method to inhibit diseases by abnormal high amount of IFP35 inducedover immune response or application of product that inhibit IFP35protein to inhibit inflammation factor expression/reaction.

Embodiment 7

The method of Embodiment 6, wherein said inhibitory product or reagentis a IFP35 antibody that inhibits IFP35 protein activity, and/or whereinthe antibody is monoclonal or polyclonal, and/or wherein said disease issepsis, and/or wherein said inflammation factors include: IL-1β, TNF-α,iNOS, or CD86.

Embodiment 8

Use of a truncated NMI protein in preparing reagents/products to provokeimmune response and inflammation or in preparing anti-tumor products,wherein said truncated NMI is truncated human NMI or mouse NMI, whereinsaid truncated human NMI protein has a sequence set forth in SEQ ID NO:8, from residues 155-240, and said truncated mouse NMI protein has asequence set forth in SEQ ID NO: 6, from residues 151-250.

Embodiment 9

A method in preparing products to provoke immune response/inflammationby NMI protein or in preparing products to against tumor/cancer, whereinsaid NMI protein is human or mouse NMI protein, said sequence of humanNMI is set forth in SEQ ID NO: 8, or said sequence of mouse IFP35 is setforth in SEQ ID NO: 6.

Embodiment 10

A method in preparing products to provoke immune response/inflammationby NMI mutant proteins or in preparing products to against tumor/cancer,wherein said NMI mutant protein is that involved residues are mutated,leading to no binding of NMI to NMI receptor or inhibitory antibody.

Embodiment 11

The method according to any of Embodiments 8-11, wherein said provokingthe immune response/inflammation stimulates inflammation factorsexpression and/or stimulates NF-κB pathway signaling, wherein saidinflammation factors include: IL-1β, TNF-α, iNOS, or CD86, and whereinsaid stimulating NF-κB pathway is to increase IκBα to reduce the totalIκBα amount.

Embodiment 12

A method of preparing a product that inhibits NMI protein activity totreat diseases induced by abnormal high expression of NMI or a productthat inhibits NMI to inhibit inflammation factors expression.

Embodiment 13

The method according to Embodiment 12, wherein said product is a NMIantibody, optionally wherein said antibody is a monoclonal or polyclonalantibody, optionally wherein said disease is sepsis, optionally whereinsaid inflammation factors include: IL-1β, TNF-α, iNOS, or CD86.

Embodiment 14

A method to over-express and crystallize truncated IFP35 protein,comprising:

1) Using bacteria cells to express the truncated protein as said inEmbodiment 1;

2) Purifying said truncated IFP35 protein;

3) Crystallizing the purified truncated protein.

Embodiment 15

The method according to Embodiment 14, further comprising: cloning thetruncated IFP35 NID fragment genes to a vector (e.g., pGEX-6p-1 vector)in order to express a N terminal GST-tagged fusion protein(GST-IFP35-NID), then transforming this plasmid vector to an E. colicell, after IPTG induction, then collecting bacteria for purification,wherein said purification step comprises: an affinity chromatography(glutathione column) step, removing of GST fusion tag by enzyme, and/oran ion exchange column step, and/or a gel filtration column step.

Embodiment 16

A method to crystallize NMI protein or its truncations, comprising:

1) Expressing the NMI truncation proteins described as in Embodiment 8in eukaryotic cells;

2) Purifying these truncated proteins;

3) Crystallizing these proteins.

Embodiment 17

The method according to Embodiment 14, further comprising: cloning thetruncated NMI NID fragment genes to a vector (e.g., pGEX-6p-1 vector) inorder to express a N terminal GST-tagged fusion protein (GST-NMI-NID),then transforming this plasmid vector to an E. coli cell, after IPTGinduction, then collecting bacteria for purification, wherein saidpurification step comprises: an affinity chromatography (glutathionecolumn) step, removing of GST fusion tag by enzyme, and/or an ionexchange column step, and/or a gel filtration column step.

Embodiment 18

A method of designing or modifying a IFP35 family protein, such as IFP35and NMI, in order to treat tumor, immune deficient diseases, etc., bytargeting the IFP35 family protein to treat the disease.

Embodiment 19

A structure having a root mean square deviation (RMSD) in its coreregion less than about 1.5 anstrom to the IFP35-NID structure disclosedherein, according to the atom coordinates of the IFP35-NID crystalstructure disclosed herein.

Embodiment 20

An atomic level structure having a root mean square deviation (RMSD)less than 1.5 astrom to the core region of IFP35-NID domain (includesresidues 136-216).

Embodiment 21

A IFP35 antibody, 1D7, produced against human IFP35 in mouse, anddeposited in China General Microbiological Culture Collection Center(CGMCC) under CGMCC 9573.

Embodiment 22

A IFP35 truncation protein having a human or mouse origin, wherein saidhuman IFP35 truncation is:

1) A protein with sequence as shown in SEQ ID NO: 4, from residues134-216; or

2) A protein with sequences same to 1) but with additional 1-10 residuesat its amino or carboxyl terminus, wherein the homology of the proteinto the protein in 1) is at least about 90%.

Embodiment 23

A IFP35 family protein having a mutation at a residue involved inbinding of the IFP35 family protein to a receptor or a neutralizingantibody, wherein the mutation disrupts or reduces binding to thereceptor or neutralizing antibody, wherein the residue is: Ser145,Asp172, Val 173, Leu 177, Arg212, Gln199, Gln207, Gln 208, Pro210,Ser214, Thr201, Tyr216, Glu150, Glu175, Gln208, Arg147, Glu151, or/andGly206 of human IFP set forth in SEQ ID NO: 2, or a residue in the IFP35family protein that corresponds to the listed residue of SEQ ID NO: 2.

Embodiment 25

A NMI truncation protein from residues 155-240 (for human NMI set forthin SEQ ID NO: 8) and from residues 151-250 (for mouse NMI forth in SEQID NO: 6).

D, Pharmaceutical Compositions for Treating and/or Preventing a Diseaseor Disorder Associated with Abnormally High Level and/or Activity ofIFP35 and/or NMI

In another aspect, the present disclosure provides for a pharmaceuticalcomposition for treating and/or preventing a disease or disorderassociated with abnormally high level of IFP35 and/or NMI in a subject,which pharmaceutical composition comprises an effective amount of anagent that prevents or reduces production and/or an activity of IFP35and/or NMI in a subject and a pharmaceutically acceptable carrier orexcipient.

The present pharmaceutical compositions can be used to treat and/orprevent any suitable disease or disorder associated with abnormally highlevel of IFP35 and/or NMI in a subject.

In some embodiments, the present pharmaceutical compositions can be usedto treat a disease or disorder associated with abnormally high level ofIFP35 and/or NMI. In other embodiments, the present pharmaceuticalcompositions can be used to prevent a disease or disorder associatedwith abnormally high level of IFP35 and/or NMI.

Any suitable agents can be used in the present pharmaceuticalcompositions to treat and/or prevent any suitable disease or disorderassociated with abnormally high level of IFP35 and/or NMI in a subject.For example, a suitable agent can be used to reduce copy number of IFP35and/or NMI gene, to block or reduce replication of IFP35 and/or NMIgene, to block or reduce transcription of IFP35 and/or NMI gene, toblock or reduce translation of IFP35 and/or NMI mRNA, and/or to inhibitone, some or all activities of IFP35 and/or NMI. In some embodiments,the agent comprises a polynucleotide (such as an siRNA, shRNA, or miRNA)targeting the gene encoding IFP35 and/or NMI. The siRNA can target anysuitable portion of the IFP35 and/or NMI gene. For example, the siRNAcan target a portion of the IFP35 and/or NMI gene that encodes one ormore NIDs. In other embodiments, the agent comprises an antisense RNAtargeting the gene encoding IFP35 and/or NMI. The antisense RNA cantarget any suitable portion of the IFP35 and/or NMI gene.

In still other embodiments, the agent inhibits or modifies a nuclearfactor that controls IFP35 and/or NMI transcription. Any suitable agentthat inhibits or modifies a nuclear factor that controls IFP35 and/orNMI transcription can be used in the present methods.

In yet other embodiments, the agent comprises an antibody thatspecifically binds to IFP35 and/or NMI. The antibody can specificallybind to any suitable portion of the IFP35 and/or NMI. For example, theantibody can specifically bind to one or more NIDs of IFP35 and/or NMI.In another example, the antibody can specifically bind to a portion ofIFP35 and/or NMI that interacts with the cellular IFP35 and/or NMIreceptors.

In some aspects, disclosed herein is an antibody or antigen bindingfragment thereof that specifically binds to IFP35 and/or NMI, and theantibody or antigen binding fragment thereof comprises a heavy chainvariable region comprising a complementarity determining region (CDR)consisting of the amino acid sequences of a CDR in the heavy chainvariable region sequence set forth in SEQ ID NO: 9 and/or a light chainvariable region comprising a CDR consisting of the amino acid sequenceof a CDR in the light chain variable region sequence set forth in SEQ IDNO: 10. In one embodiment, the antibody or antigen binding fragmentthereof comprises a heavy chain variable region set forth in SEQ ID NO:9 and a light chain variable region set forth in SEQ ID NO: 10.Conservative substitution(s) may be introduced into the CDRs and/orframework regions (FRs) of an antibody disclosed herein.

(SEQ ID NO: 9) V Q L V E S G P E L K K P G E T V K I S C K A SG Y T F T N Y G M N W V K Q A P G K G L K W M GW I N T Y T G E P T F A D D F K G R F A F S L ET S A S T A Y L Q I N N L K N E D T A T Y F C AR Y G Y S W A M D Y W G Q G T S V T V S S A S T. (SEQ ID NO: 10)D I V M T Q S P A I M S A S P G E K V T M T C SA S S S V S Y M H W Y Q Q K S G T S P K R W I YD T S K L A S G V P A R F S G S G S G T S Y S LT I S S M E A E D A A T Y Y C Q Q W S S N P P I T F G A G T K L E I K.

In one aspect, disclosed herein is an isolated polynucleotide encodingan antibody or antigen binding fragment thereof that specifically bindsto IFP35 and/or NMI. In some embodiments, the antibody or antigenbinding fragment thereof comprises a heavy chain variable regioncomprising a complementarity determining region (CDR) consisting of theamino acid sequence of a CDR in the heavy chain variable region sequenceset forth in SEQ ID NO: 9 and/or a light chain variable regioncomprising a CDR consisting of the amino acid sequences of a CDR in thelight chain variable region sequence set forth in SEQ ID NO: 10. In someaspects, the antibody or antigen binding fragment thereof comprises aheavy chain variable region set forth in SEQ ID NO: 9 and a light chainvariable region set forth in SEQ ID NO: 10. In other embodiments, theisolated polynucleotide comprises the nucleic acid sequence sent forthin SEQ ID NO: 11 and/or SEQ ID NO: 12.

(SEQ ID NO: 11) TGGTCGACGCTGAGGAGACGGTGACTGAGGTTCCTTGACCCCAGTAGTCCATAGCCCAAGAGTACCCGTATCTTGCACAGAAATATGTAGCCGTGTCCTCATTCTTGAGGTTGTTGATCTGCAAATAGGCAGTGCTGGCAGAGGTTTCCAAAGAGAAGGCAAACCGTCCCTTGAAGTCATCAGCAAATGTTGGCTCTCCAGTGTAGGTGTTTATCCAGCCCATCCACTTTAAACCCTTTCCTGGAGCCTGCTTCACCCAGTTCATTCCATAGTTTGTGAAGGTATACCCAGAAGCCTTGCAGGAGATCTTGACTGTCTCTCCTGACTCCTTAAGCT GCACCT. (SEQ ID NO: 12)GACATTGTGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACCCATCACGTTCGGTGCTGGCACCAAGCTGGAAATCAAA.

The terms “complementarity determining region,” and “CDR,” are known inthe art to refer to non-contiguous sequences of amino acids withinantibody variable regions, which confer antigen specificity and bindingaffinity. In general, there are three (3) CDRs in each heavy chainvariable region (CDR-H1, CDR-H2, CDR-H3) and three (3) CDRs in eachlight chain variable region (CDR-L1, CDR-L2, CDR-L3).

The precise amino acid sequence boundaries of a given CDR can be readilydetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numberingscheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996),“Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745.” (Contact” numbering scheme),Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains,” Dev CompImmunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme), andHonegger A and Plückthun A, “Yet another numbering scheme forimmunoglobulin variable domains: an automatic modeling and analysistool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme).

The boundaries of a given CDR may vary depending on the scheme used foridentification. For example, the Kabat scheme is based structuralalignments, while the Chothia scheme is based on structural information.Numbering for both the Kabat and Chothia schemes is based upon the mostcommon antibody region sequence lengths, with insertions accommodated byinsertion letters, for example, “30a,” and deletions appearing in someantibodies. The two schemes place certain insertions and deletions(“indels”) at different positions, resulting in differential numbering.The Contact scheme is based on analysis of complex crystal structuresand is similar in many respects to the Chothia numbering scheme.

Thus, unless otherwise specified, the terms “CDR” and “complementarydetermining region” of a given antibody or region thereof, such as avariable region, as well as individual CDRs (e.g., “CDR-H1, CDR-H2) ofthe antibody or region thereof, should be understood to encompass thecomplementary determining region as defined by any of the known schemesdescribed herein above.

As used herein, the term “conservative substitution” refers tosubstitutions of amino acids and/or amino acid sequences that are knownto those of skill in this art and may be made generally without alteringthe biological activity of the resulting molecule. Those of skill inthis art recognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson, et al., MOLECULAR BIOLOGY OF THEGENE, The Benjamin/Cummings Pub. Co., p. 224 (4th Edition 1987)). Forexample, such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments (see, pages 13-15“Biochemistry” 2nd ED. Lubert Stryer ed (Stanford University); Henikoffet al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May19; 270(20):11882-6). Other substitutions are also permissible and maybe determined empirically or in accord with known conservativesubstitutions.

In yet other embodiments, the agent can comprise a variant form of IFP35and/or NMI. Any suitable variant form of IFP35 and/or NMI can be used inthe present methods. For example, the variant form of IFP35 and/or NMIcan be a mutant IFP35 and/or NMI and/or a fragment of IFP35 and/or NMI.

The pharmaceutical compositions can be administered via any suitableroute. For example, the pharmaceutical compositions can be administeredvia an oral, nasal, inhalational, parental, intravenous,intraperitoneal, subcutaneous, intramuscular, intradermal, topical, orrectal route.

In some embodiments, the present pharmaceutical compositions can furthercomprise another suitable therapeutic or preventive substance ortreatment. For example, the pharmaceutical compositions can furthercomprise an effective amount of a drug for the treatment and/orprevention of a proliferation disorder, a neoplasm, a tumor or a cancerin the subject.

The present pharmaceutical compositions can be used at any suitableamount or dosage. For example, the present pharmaceutical compositionscan be administered at an amount that reduces production and/or anactivity of IFP35 and/or NMI in the subject to a level that issubstantially identical to a production and/or an activity level ofIFP35 and/or NMI in a comparable subject that does not have a disease ordisorder associated with abnormally high level of IFP35 and/or NMI.

The present pharmaceutical compositions can be used to treat and/orprevent a disease or disorder in any suitable subject. For example, thesubject can be a mammal. In some embodiments, the mammal is a human. Inother embodiments, the subject is a non-human animal or non-humanmammal, e.g., experimental, companion or economic non-human animal ornon-human mammal.

E, Use of an Effective Amount of an Agent that Prevents or ReducesProduction and/or an Activity of IFP35 and/or NMI in a Subject for theManufacture of a Medicament

In still another aspect, the present disclosure provides for a use of aneffective amount of an agent that prevents or reduces production and/oran activity of IFP35 and/or NMI in a subject for the manufacture of amedicament for treating and/or preventing a disease or disorderassociated with abnormally high level of IFP35 and/or NMI in saidsubject.

An agent that prevents or reduces production and/or an activity of IFP35and/or NMI in a subject can be used for the manufacture of a medicamentfor treating and/or preventing any suitable disease or disorderassociated with abnormally high level of IFP35 and/or NMI in saidsubject.

In some embodiments, agent that prevents or reduces production and/or anactivity of IFP35 and/or NMI in a subject can be used for themanufacture of a medicament for treating any suitable disease ordisorder associated with abnormally high level of IFP35 and/or NMI insaid subject. In other embodiments, agent that prevents or reducesproduction and/or an activity of IFP35 and/or NMI in a subject can beused for the manufacture of a medicament for preventing any suitabledisease or disorder associated with abnormally high level of IFP35and/or NMI in said subject.

Any suitable agents can be used for the manufacture of a medicament fortreating any suitable disease or disorder associated with abnormallyhigh level of IFP35 and/or NMI in a subject. For example, a suitableagent can be used to reduce copy number of IFP35 and/or NMI gene, toblock or reduce replication of IFP35 and/or NMI gene, to block or reducetranscription of IFP35 and/or NMI gene, to block or reduce translationof IFP35 and/or NMI mRNA, and/or to inhibit one, some or all activitiesof IFP35 and/or NMI. In some embodiments, the agent comprises an siRNAtargeting the gene encoding IFP35 and/or NMI. The siRNA can target anysuitable portion of the IFP35 and/or NMI gene. For example, the siRNAcan target a portion of the IFP35 and/or NMI gene that encodes one ormore NID domains.

In other embodiments, the agent comprises an antisense RNA targeting thegene encoding IFP35 and/or NMI. The antisense RNA can target anysuitable portion of the IFP35 and/or NMI gene. For example, theantisense RNA can target a portion of the IFP35 and/or NMI gene thatencodes one or more NID domains.

In still other embodiments, the agent inhibits or modifies a nuclearfactor that controls IFP35 and/or NMI transcription. Any suitable agentthat inhibits or modifies a nuclear factor that controls IFP35 and/orNMI transcription can be used for the manufacture of a medicament fortreating and/or preventing a disease or disorder associated withabnormally high level of IFP35 and/or NMI in a subject.

In yet other embodiments, the agent comprises an antibody thatspecifically binds to IFP35 and/or NMI. The antibody can specificallybind to any suitable portion of the IFP35 and/or NMI. For example, theantibody can specifically bind to one or more NID domains of IFP35and/or NMI. In another example, the antibody can specifically bind to aportion of a NID domain of IFP35 and/or NMI.

In yet other embodiments, the agent can comprise a variant form of IFP35and/or NMI. Any suitable variant form of IFP35 and/or NMI can be used inthe present methods. For example, the variant form of IFP35 and/or NMIcan be a mutant IFP35 and/or NMI and/or a fragment of IFP35 and/or NMI,e.g., a fragment comprising one or more NID domains of IFP35 and/or NMI,or a fragment comprising a portion of a NID domain of IFP35 and/or NMI.The agent can also comprise fusion proteins between a variant form ofIFP35 and/or NMI and a heterologous protein (such as an Fc portion).

The medicament can be manufactured for administration via any suitableroute. For example, the medicament can be administered via an oral,nasal, inhalational, parental, intravenous, intraperitoneal,subcutaneous, intramuscular, intradermal, topical, or rectal route.

In some embodiments, the present medicaments can further compriseanother suitable therapeutic or preventive substance or treatment. Forexample, the medicaments can further comprise an effective amount of adrug for the treatment and/or prevention of a proliferation disorder, aneoplasm, a tumor or a cancer in the subject.

The present medicaments can be used at any suitable amount or dosage.For example, the present medicaments can be administered at an amountthat reduces production and/or an activity of IFP35 and/or NMI in thesubject to a level that is substantially identical to a productionand/or an activity level of IFP35 and/or NMI in a comparable subjectthat does not have a disease or disorder associated with abnormally highlevel of IFP35 and/or NMI.

The present medicaments can be used to treat and/or prevent a disease ordisorder in any suitable subject. For example, the subject can be amammal. In some embodiments, the mammal is a human. In otherembodiments, the subject is a non-human animal or non-human mammal,e.g., experimental, companion or economic non-human animal or non-humanmammal.

Any suitable formulation of the compounds described herein can beprepared. See, generally, Remington's Pharmaceutical Sciences, (2000)Hoover, J. E. editor, 20th edition, Lippincott Williams and WilkinsPublishing Company, Easton, Pa., pages 780-857. A formulation isselected to be suitable for an appropriate route of administration. Someroutes of administration are oral, parenteral, by inhalation, topical,rectal, nasal, buccal, vaginal, via an implanted reservoir, or otherdrug administration methods. In cases where compounds are sufficientlybasic or acidic to form stable nontoxic acid or base salts,administration of the compounds as salts may be appropriate. Examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids that form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceuticallyacceptable salts are obtained using standard procedures well known inthe art, for example, by a sufficiently basic compound such as an aminewith a suitable acid, affording a physiologically acceptable anion.Alkali metal (e.g., sodium, potassium or lithium) or alkaline earthmetal (e.g., calcium) salts of carboxylic acids also are made.

Where contemplated compounds are administered in a pharmacologicalcomposition, it is contemplated that the compounds can be formulated inadmixture with a pharmaceutically acceptable excipient and/or carrier.For example, contemplated compounds can be administered orally asneutral compounds or as pharmaceutically acceptable salts, orintravenously in a physiological saline solution. Conventional bufferssuch as phosphates, bicarbonates or citrates can be used for thispurpose. Of course, one of ordinary skill in the art may modify theformulations within the teachings of the specification to providenumerous formulations for a particular route of administration. Inparticular, contemplated compounds may be modified to render them moresoluble in water or other vehicle, which for example, may be easilyaccomplished with minor modifications (salt formulation, esterification,etc.) that are well within the ordinary skill in the art. It is alsowell within the ordinary skill of the art to modify the route ofadministration and dosage regimen of a particular compound in order tomanage the pharmacokinetics of the present compounds for maximumbeneficial effect in a patient.

The agents as described herein can be or can be made generally solublein organic solvents such as chloroform, dichloromethane, ethyl acetate,ethanol, methanol, isopropanol, acetonitrile, glycerol,N,N-dimethylformamide, N,N-dimetheylaceatmide, dimethylsulfoxide, etc.In one embodiment, the present invention provides formulations preparedby mixing an agent with a pharmaceutically acceptable carrier. In oneaspect, the formulation may be prepared using a method comprising: a)dissolving a described agent in a water-soluble organic solvent, anon-ionic solvent, a water-soluble lipid, a cyclodextrin, a vitamin suchas tocopherol, a fatty acid, a fatty acid ester, a phospholipid, or acombination thereof, to provide a solution; and b) adding saline or abuffer containing 1-10% carbohydrate solution. In one example, thecarbohydrate comprises dextrose. The pharmaceutical compositionsobtained using the present methods are stable and useful for animal andclinical applications.

Illustrative examples of water soluble organic solvents for use in thepresent methods and compositions include and are not limited topolyethylene glycol (PEG), alcohols, acetonitrile,N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide,dimethyl sulfoxide, or a combination thereof. Examples of alcoholsinclude but are not limited to methanol, ethanol, isopropanol, glycerol,or propylene glycol.

Illustrative examples of water soluble non-ionic surfactants for use inthe present methods and compositions include and are not limited toCREMOPHOR® EL, polyethylene glycol modified CREMOPHOR®(polyoxyethyleneglyceroltriricinoleat 35), hydrogenated CREMOPHOR® RH40,hydrogenated CREMOPHOR® RH60, PEG-succinate, polysorbate 20, polysorbate80, SOLUTOL® HS (polyethylene glycol 660 12-hydroxystearate), sorbitanmonooleate, poloxamer, LABRAFIL® (ethoxylated persic oil), LABRASOL®(capryl-caproyl macrogol-8-glyceride), GELUCIRE® (glycerol ester),SOFTIGEN® (PEG 6 caprylic glyceride), glycerin, glycol-polysorbate, or acombination thereof.

Illustrative examples of water soluble lipids for use in the presentmethods and compositions include but are not limited to vegetable oils,triglycerides, plant oils, or a combination thereof. Examples of lipidoils include but are not limited to castor oil, polyoxyl castor oil,corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil,safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil,hydrogenated soybean oil, a triglyceride of coconut oil, palm seed oil,and hydrogenated forms thereof, or a combination thereof.

Illustrative examples of fatty acids and fatty acid esters for use inthe present methods and compositions include but are not limited tooleic acid, monoglycerides, diglycerides, a mono- or di-fatty acid esterof PEG, or a combination thereof.

Illustrative examples of cyclodextrins for use in the present methodsand compositions include but are not limited to alpha-cyclodextrin,beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, or sulfobutylether-beta-cyclodextrin.

Illustrative examples of phospholipids for use in the present methodscyclodextrin include but are not limited to soy phosphatidylcholine, ordistearoyl phosphatidylglycerol, and hydrogenated forms thereof, or acombination thereof.

One of ordinary skill in the art may modify the formulations within theteachings of the specification to provide numerous formulations for aparticular route of administration. In particular, the compounds may bemodified to render them more soluble in water or other vehicle. It isalso well within the ordinary skill of the art to modify the route ofadministration and dosage regimen of a particular compound in order tomanage the pharmacokinetics of the present compounds for maximumbeneficial effect in a patient.

F, Method of Diagnosis, Prognosis or Treatment Monitoring

In yet another aspect, the present disclosure provides for a method ofdiagnosis, prognosis or treatment monitoring of a disease or disorderassociated with abnormally high level of IFP35 and/or NMI in a subject,which method comprises assessing the level and/or an activity of IFP35and/or NMI in a subject suspected of or being treated for a disease ordisorder associated with abnormally high level of IFP35 and/or NMI.

The present methods can be used for diagnosis, prognosis or treatmentmonitoring of any suitable disease or disorder associated withabnormally high level of IFP35 and/or NMI in a subject.

In some embodiments, the present methods are used for diagnosis of adisease or disorder associated with abnormally high level of IFP35and/or NMI in a subject, and wherein a level and/or an activity of IFP35and/or NMI in a subject that is at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% higher than a leveland/or an activity of IFP35 and/or NMI in a comparable subject that doesnot have a disease or disorder associated with abnormally high level ofIFP35 and/or NMI, e.g., a proliferation disorder, a neoplasm, a tumor ora cancer, indicates that the subject has the disease or disorderassociated with abnormally high level of IFP35 and/or NMI.

In some embodiments, the present methods are used for treatmentmonitoring of a disease or disorder associated with abnormally highlevel of IFP35 and/or NMI in a subject to adjust further treatment ofthe subject, e.g., to increase or decrease the dosage, or to extend,shorten or stop the treatment.

In some embodiments, the present methods comprise assessing the level ofIFP35 and/or NMI in a subject suspected of or being treated for adisease or disorder associated with abnormally high level of IFP35and/or NMI. In other embodiments, the present methods comprise assessingan activity of IFP35 and/or NMI in a subject suspected of or beingtreated for a disease or disorder associated with abnormally high levelof IFP35 and/or NMI. Any suitable IFP35 and/or NMI activity can beassessed, e.g., binding to a cellular receptor or an antibody. In stillother embodiments, the present methods comprise assessing the level andan activity of IFP35 and/or NMI in a subject suspected of or beingtreated for a disease or disorder associated with abnormally high levelof IFP35 and/or NMI.

The level and/or an activity of IFP35 and/or NMI can be assessed at anysuitable level. For example, the level and/or an activity of IFP35and/or NMI can be assessed at the DNA, RNA and/or protein level. Thelevel of IFP35 and/or NMI DNA and/or RNA can be assessed using anysuitable means or methods. For example, the level of IFP35 and/or NMIDNA and/or RNA can be assessed using a polynucleotide that iscomplementary to at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more consecutivenucleotides in the IFP35 and/or NMI DNA or RNA. The level of IFP35and/or NMI protein can be assessed using any suitable means or methods.For example, the level of IFP35 and/or NMI protein can be assessed usingan antibody that specifically binds to IFP35 and/or NMI. Any suitableantibody can be used. For example, the antibody can specifically bind toa portion of a NID domain, or one or more NID domains of IFP35 and/orNMI. In still another example, the antibody is a polyclonal antibody.

In yet another aspect, the present disclosure provides for a kit ofdiagnosis, prognosis or treatment monitoring of a disease or disorderassociated with abnormally high level of IFP35 and/or NMI in a subject,which kit comprises means for assessing the level and/or an activity ofIFP35 and/or NMI in a subject suspected of or being treated for adisease or disorder associated with abnormally high level of IFP35and/or NMI.

Any suitable means can be used in the present kits. For example, themeans can be used in assessing an activity of IFP35 and/or NMI. Inanother example, the means can be a polynucleotide that is complementaryto at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1,000 or more consecutive nucleotides in theIFP35 and/or NMI DNA or RNA for assessing the level of IFP35 and/or NMIDNA and/or RNA. In still another example, the means can be an antibodythat specifically binds to IFP35 and/or NMI for assessing the level of1FP35 and/or NMI protein. The suitable means can be contained in anysuitable single or multiple containers, e.g., test tubes, microtiterplates, etc. The suitable means can also be immobilized on any suitablesingle or multiple surfaces, e.g., beads, chips or microfluidic devices,etc.

In yet another aspect, the present disclosure provides for a method ofcompanion diagnostics of a disease or disorder associated withabnormally high level of IFP35 and/or NMI in a subject, which methodcomprises determining the genetic status of IFP35 and/or NMI gene in asubject being treated for the disease or disorder associated withabnormally high level of IFP35 and/or NMI.

The genetic status of IFP35 and/or NMI gene in a subject can be assessedusing any suitable methods or means. For example, the genetic status ofIFP35 and/or NMI gene in a subject can be determined using apolynucleotide that is complementary to at least 10, 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000consecutive nucleotides in the IFP35 and/or NMI DNA or RNA.

In yet another aspect, the present disclosure provides for a kit ofcompanion diagnostics of a disease or disorder associated withabnormally high level of IFP35 and/or NMI in a subject, which kitcomprises means for determining the genetic status of IFP35 and/or NMIgene in a subject being treated for the disease or disorder associatedwith abnormally high level of IFP35 and/or NMI.

Any suitable means can be used in the present kits. For example, thepresent kits can comprise a polynucleotide that is complementary to atleast 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1,000 consecutive nucleotides in the IFP35 and/orNMI DNA or RNA for determining the genetic status of IFP35 and/or NMIgene in a subject. The suitable means can be contained in any suitablesingle or multiple containers, e.g., test tubes, microtiter plates, etc.The suitable means can also be immobilized on any suitable single ormultiple surfaces, e.g., beads, chips or microfluidic devices, etc.

The present methods can be conducted in any suitable manner. Forexample, the present methods can be conducted manually. In anotherexample, the present methods can be conducted in a semi-automatic or anautomatic fashion. The present methods can be conducted at any suitablelocation. For example, the present methods can be conducted at ahospital, a clinic, a doctor's office, a clinical lab, a pharmacy, anoffice or a home. The present methods can be conducted at by anysuitable personnel. For example, the present methods can be conducted bya physician, a nurse, a lab technician, a caregiver or a patient.

The present kits can comprises additional suitable components, e.g.,means for obtaining a sample from a subject, assay standard, identifierof the test subject and/or test instructions, etc. The present kits canbe standalone kits or can be part of an assay system, e.g., automatedassay systems.

G, Methods for Identifying a Modulator of IFP35 and/or NMI

In yet another aspect, the present disclosure provides for a method foridentifying a modulator of IFP35 and/or NMI, which method comprises: a)contacting IFP35 and/or NMI with a test substance and assessing anactivity of IFP35 and/or NMI that has been contacted by said testsubstance; b) assessing an activity of said IFP35 and/or NMI that hasnot been contacted by said test substance; and c) comparing saidactivities of IFP35 and/or NMI assessed in steps a) and b), andidentifying said test substance as a modulator of IFP35 and/or NMI whensaid activities of IFP35 and/or NMI assessed in steps a) and b) aredifferent.

Any suitable test substances can be used in the present methods. Forexample, the test substances can be small molecules, a polypeptidelibrary comprising mutants and/or fragments of IFP35 and/or NMI,antibodies that specifically bind IFP35 and/or NMI, siRNAs or antisenseRNAs.

In some embodiments, the present methods can be used to identify aninhibitor of an activity of IFP35 and/or NMI.

In some embodiments, the present methods can be used to identify a drugfor treating and/or preventing a disease or disorder associated withabnormally high level of IFP35 and/or NMI in a subject. The presentmethods can be used to identify a drug for treating and/or preventingany suitable disease or disorder associated with abnormally high levelof IFP35 and/or NMI in a subject. For example, the disease or disorderassociated with abnormally high level of IFP35 and/or NMI in a subjectcan be a proliferation disorder, a neoplasm, a tumor or a cancer.

The present methods can be conducted using any suitable assay format.Preferably, the present methods can be conducted using a high-throughputassay format.

In some embodiments, the present disclosure provides for a modulator ofIFP35 and/or NMI or a drug candidate that is identified by presentmethods.

H, Examples Example 1: IFP35 Family Members as Early Endogenous DAMPsAbstract

In this example, the structures of a hybrid NID domain (NID-H) of IFP35were determined. NID-H could fold into an open or a closed conformation.NID-H in the open conformation showed high virulence to mice by inducingcytokine cascade in macrophages, characterized by the up-regulation ofthe expression of some early inflammation factors. Furthermore, IFP35and NMI were released into the cell culture media of monocytes ormacrophages, 1 hour after stimulated by LPS or IFN-γ. The accumulationof IFP35 and NMI were also detected in the serum of LPS shocked mice andseptic patients. A neutralizing antibody to IFP35 NID-H attenuated theLPS induced inflammatory response, and effectively improved the survivalrate of septic mice. Besides, IFP35 or NMI knock-out mice were resistantto LPS challenge. Compared to HMGB-1, a known late DAMP, the IFP35family members could serve as endogenous ligands of TLR4, leading to theactivation of the transcription factor NF-κB. Therefore, in someaspects, IFP35 family members with one or more NID domains, includingIFP35 and NMI, could serve as early endogenous DAMPs. In some aspects,use of IFP35 and/or NMI or binding partners in the diagnosis and/ortreatment of infection and injury is provided.

Results

1. The Structures of NID Revealed its Open and Closed Conformations.

According to sequence analysis, IFP35 has an L-zip domain at itsN-terminus containing the first 80 amino acids, following by two tandemNID domains covering the 81-170 and 177-268 amino acids, respectively(FIG. 1a ). Truncated protein constructs were constructed according tosecondary structure prediction. A truncated IFP35 protein containing the124^(th) to the 220^(th) amino acids expressed well in E. coli. Sincethis truncated IFP35 protein spreads across the two predicted NIDdomains, it was termed NID-H (heterozygous NID) (FIG. 1a ). During thepurification process, NID-H was found to exist in at least two stableaggregation states, dimer and octamer (FIG. 2a ).

Well-diffracted NID-H crystals with both dimeric and octameric stateswere obtained. The crystal of dimeric NID-H with seleno-L-methioninederivation diffracted at 2.2 Å. The structure was determined bysingle-wavelength anomalous diffraction (SAD) method (Table 1). The SADmethod is disclosed in Chayen & Saridakis, Nature methods 5, 147-153(2008). The NID-H folds into barrel-like structure, formed by fiveβ-strands and two α-helixes which connect with each other in the orderof β1-α1-β2-β3-α2-β4-β5 (FIG. 1b ). Several pairs of residues on helixα1 and the following loop (L1) connecting α1 and β2 play important rolesfor dimer formation. Based on the structure, at least five pairs ofinteractions could be observed (FIG. 1c ), including: the hydrophobicinteraction between the side chains of Ile159 from each monomer; theelectronic interactions between Lys163 and Glu158, as well as Asp155 andLys156; the interaction between the side chain of Asp170 and the aminogroup on the backbone of Thr164; the interaction between the side chainof Arg165 and carbonyl group on the backbone of Val171.

TABLE 1 Data collection and refinement statistics Dimer (Se-Met) Octamer(Native) Data Collection Beamline BL17U1 BL17U1 Wavelength (Å) 0.97980.9792 Resolution range (Å) 40.0-2.3 (2.34-2.30)^(a) 50.0-2.5(2.54-2.50) Space group H3 P2₁ Cell dimensions a, b, c (Å) 170.3, 170.3,53.0 44.4, 125.3, 77.2 α, β, γ (°) 90.0, 90.0, 120.0 90.0, 90.3, 90.0Total reflections 25,256 (1,263) 29,326 (1,447) Unique reflections 1,1486,110 Completeness (%) 99.9 (100.0) 97.8 (99.9) Mean I/σ 74.2 (21.3)21.1 (5.4) Multiplicity 22.0 (22.5) 4.8 (5.4) R_(sys) (%) 12.5 (69.9)10.8 (57.9) Refinement Resolution range (Å) 32.38-2.30 36.71-2.50R_(work)/R_(free) (%) 18.1/22.9 20.6/25.3 Rmsd bond length (Å) 0.0020.003 Rmsd bond angles (°) 0.619 0.639 Ramachandran plot Most favored(%) 98.2 97.6 Additional allowed (%) 1.8 2.4 Disallowed (%) 0.0 0.0Average B factor (Å²) 29.0 55.0 ^(a)Values in parentheses are for thehighest resolution shell.

Subsequently, the octameric structure of NID-H was determined at 2.8 Åresolution by molecular replacement (MR) method (FIG. 1d , Table 1). Adomain-swapping conformation between two monomers can be observed in theoctameric structure. The specific manner is that β1-α1-β2 from onemonomer interact with β3-α2-β4-β5 from another monomer and fold into abarrel-like structure (FIG. 1d ). In this way, two monomers piled uphead-to-end and interact tightly in the dimer. For clarity, thisdomain-swapping dimer was named the open conformational dimer (o-dimer).The dimer without domain-swapping is called the closed conformationaldimer (c-dimer). Four o-dimers further interact with each other throughα1-α1 interactions, as described in c-dimer, and form a ring likestructure (FIG. 2b ). The monomer in o-dimer is quite similar to that inthe c-dimer (FIG. 1e ). The root mean square deviation (RMSD) betweenthem is only about 0.7 Å. The major difference is that the β-turn (Ti)in c-dimer connecting β2 and β3 is straightened in the o-dimer, in whichit forms a loop (L2) and connects the two barrel-like structures. Thecrystal structure coordinates of the dimer structure and the octamerstructure are provided in FIG. 33 and FIG. 34, respectively.

Using Dali server, a powerful structure comparison tool, knownstructures in PDB were compared to the barrel-like structure of NID-H.The NID-H structure was found to be very similar to that of RNArecognition motif (RRM) (FIG. 2c ). The RRMs are widely used to identifyRNAs of different sequences and structures. However, there is no obviousRNA binding site in the structure of NID-H indicated by its surfacecharge distribution. EMSA experiment using NID-H and RNAs with differentsequences were also performed, and no interaction between NID-H and thetested RNA sequences was found (data not shown).

2. The Open Conformation of IFP35 NID-H Stimulated Immune Response.

NID-H domain was used as antigen to stimulate polyclonal antibodiesproduction in mice for IFP35 detection. The NID-H octamer caused a 100%mortality rate for all four mice used. Besides, all the mice died withswelled belly and obviously increased ascites. This phenomenon indicatedthat the NID-H octamer might have cellular toxicity. Because IFP35typically is specifically expressed in immune cells, such as monocytes,macrophages, dendritic cells and lymphocytes, the NID-H octamer couldstimulate inflammatory response.

The transcription level of pro-inflammatory factors, such as TNF-α andIL-1β, were up-regulated in the presence of NID-H octamer in murinemacrophage-like RAW264.7 cells (FIG. 3a ). This result was similar tolipopolysaccharide (LPS) induced inflammatory cytokines production. Bycomparison, the NID-H dimer (c-dimer) did not have this effect. Based onthese results, NID-H in its octameric structure tends to induce cytokinestorm related to TNF-α and IL-1β.

Since the c-dimer itself could not induce inflammation, the o-dimer inthe NID-H octamer could serve as the active conformation. To verifywhether the octameric skeleton is necessary for its cellular toxicity, amutant with three point mutations, K156E/K163E/R165E, on the α1 helix asshown in FIG. 1c , was constructed. These mutations were supposed topartially destroy the c-dimer formation, which leads to thede-polymerization of the octamer. Therefore, some independent o-dimershould be produced. The results showed that the mutated and/or modifieddimer could partially up-regulate the transcription of TNF-α and IL-1β(FIG. 3b ), which could be ascribed to the formation of independento-dimers. The results showed that o-dimer is the actitive state of NID-Hto induce cytokine storm.

Since NID-H can activate the innate immune response, IFP35 full lengthprotein was expressed. However, IFP35 had low yield in eukaryotic cellsand formed inclusion body in E. coli., probably due to the hydrophobicL-zip domain at its N terminus. A truncated IFP35 with 34 amino acidsdeletion at its N terminal (ΔN) was constructed and expressed usingprokaryotic expression system. Soluble ΔN protein was obtained, and theΔN protein stimulated the transcription of TNF-α and IL-1β moreeffectively than NID-H octamer (FIG. 3c ). Compared to the effectivedose of NID-H (about 10-50 μg/ml), the effective dose of ΔN was about 1μg/ml, which was comparable to that of HMGB-1 or Mrps. Besides, ΔN has astrong effects to stimulate the release of inflammation cytokines suchas TNF-α at low dose. Detected by ELISA, 100-200 μg/ml TNF-α could bereleased into culture by RAW 264.7 cells, stimulated by ΔN or LPS at 1μg/ml. (FIG. 3d ). Moreover, when RAW264.7 cells were stimulated usingrecombinant mNMI protein, similar inflammation response was alsodetected (FIG. 4). Based on these results, IFP35 family members canserve as endogenous DAMP proteins.

A secondary structure prediction of human IFP35 is shown in FIG. 32.According to the prediction, in some aspects, removal of the first 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, or 78 residues in human IFP35 would produce similar resultsas ΔN used in this example.

3. IFP35 Family Members Possess the Typical Characteristics ofEndogenous DAMP.

DAMPs, including HSPs, HMGB-1, Mrp8 and Mrp14, are secreted by cells andare then recognized by the pattern recognition receptors (PRRs) on thecell surface. Kawai et al., Immunity 34, 637-650 (2011); Wu & Chen,Annual Review of Immunology 32, 461-488 (2014); and Zhong et al.,Frontiers in Immunology 4, 333 (2013). IFP35 and NMI were reported tolocalize in cytoplasm after being up-regulated by IFN-γ. However, inthis example, with in 3 hour after stimulated by IFN-γ, or within 1 hourafter stimulated by LPS or Salmonella, IFP35 and NMI were secreted anddetected in the culture supernatant of RAW 264.7 cells (FIG. 5a , FIG.6a , FIG. 6b and FIGS. 18-21). In the following several hours, IFP35 andNMI accumulated in the culture in a time-dependent manner as revealed byimmunoblot result. In contrast, early and late mediators of endotoxemia,TNF-α and HMGB-1, were released at several minutes and at 6-8 hours,respectively. Wang et al., Science 285, 248-251 (1999); and Tracey etal., Science 234, 470-474 (1986). IFP35 was reported to bind NMI incells, forming HMMC and further into speckle-like aggregations. Chen etal., JBC 275, 36278-84 (2000). Secreted IFP35 and NMI in the culture ofRAW264.7 cells stimulated by Salmonella SR-11 were analyzed using gelfiltration and western blot. The results showed that secreted IFP35existed as monomer and NMI existed as dimer in solution (FIG. 6c andFIG. 6d ). Similar to HMGB-1 and Mrps, there is no obvious transmembranesignal peptide in the sequences of the IFP35 family members. Therefore,the IFP35 family of proteins can serve as early DAMPs.

DAMPs were reported to be closely related to the infection and injury.Hirsiger et al., Mediators of Inflammation, 315941 (2012); and Bianchi,Journal of Leukocyte Biology 81, 1-5 (2007). The concentrations of knownDAMPs, such as HMGB-1 and Mrps, are significantly increased in the serumof septic patients. Wang et al., Science 285, 248-251 (1999); Austermannet al., Cell Reports 9, 2112-2123 (2014); and Sunden-Cullberg et al.,Critical Care Medicine 33, 564-573 (2005). In order to determine ifIFP35 and NMI were released in serum during infection, mice werechallenged using LPS and detected them in the serum of septic mice.IFP35 and NMI were secreted within 3 hours after LPS exposure. Comparedwith the control group, the serum IFP35 and NMI in LPS administratedmice increased sharply with a dose dependent manner, up to about 120ng/ml within 6 hours (FIG. 5b and FIG. 6e ). In addition, serum of thepatients with sepsis was analyzed and the concentrations of IFP35 inalmost all of the 12 samples were elevated. Although great deviationsexisted among these cases, probably caused by different pathogens andphysical conditions, the serum IFP35 could be quantified at 1-10 ng/ml,while the protein was barely detectable in the serum of normalindividuals (FIG. 5c and FIG. 6f ). These results demonstrated thatIFP35 is released into serum during infection.

Different from HMGB-1, IFP35 family members could be released within thefirst hour after LPS stimulation. Administration of antibodies to NID-H(anti-NID-H) before lethal dose (LD₁₀₀) of LPS exposure could provideeffective protection to mice. Compared with the 0% survival rate incontrol, more than 60% of the anti-NID-H treated mice survived in 7 daysafter LPS exposure (FIG. 5d ). Because the toxic effects of LPS arepartly mediated by inflammatory cytokines such as TNF-α, IL-1β and IL-6,the concentrations of these cytokines in serum after the administrationof LPS and anti-IFP35 monoclonal antibody were examined. Consistent withthis phenomenon, administration of anti-NID-H could decrease the releaseof some toxic inflammatory factors in the serum of septic mice such asTNF-α, IL-1β and IL-6, by about 20%-50% (FIG. 5e ). IFP35 and NMIknock-out mice were produced using CRISPPR-Cas9 technology. Ran et al.,Nature Protocols 8, 2281-2308 (2013). The reaction to bacterial LPS inconjunction with galactosamine (D-gal) is a well-characterized model ofendotoxic shock in mice that is mediated by inflammatory cytokines. Voglet al., Nature Medicine 13, 1042-1049 (2007); and Galanos et al., PNAS76, 5939-5943 (1979). After intraperitoneal injection of LPS and D-gal,wild-type animals showed symptoms of acute illness and died within 5-8h. In contrast, IFP35^(−/−) mice were less severely affected andsurvived significantly longer (FIG. 5f ). Resistant of IFP35^(−/−) andNMI^(−/−) mice to LPS-induced lethal toxicity was also observed whenmice were injected with a large amount of LPS without D-gal. Comparedwith the normal mice whose lethality is almost 100% under administrationof lethal dose of LPS, the lethality of NMI knock-out mice are improved(FIG. 6g ). During LPS exposure, the concentrations of knownpro-inflammatory factors were greatly decreased in the serum of theseknock-out mice (FIG. 5g and FIG. 6h ). Therefore, all these resultsdemonstrated that IFP35 could serve as DAMP and cause stronginflammatory response.

4. IFP35 Stimulates the Cytokines Storm Potentially Based on the TLR4Pathway.

Based on previous reports, HMGB-1 and Mrps induce immune responsethrough triggering the activation of NF-κB by TLR4 signal pathway. Voglet al., Nature Medicine 13, 1042-1049 (2007); Yang et al., PNAS 107,11942-11947 (2010); and Yu et al., Shock 26, 174-179 (2006).Accordingly, several key players in this pathway were detected and theireffects in immune response induced by IFP35 family members wereanalyzed. First of all, the activation of NF-κB promoter by NID-H wasdetected using luciferase assay. HEK293 cells were transientlytransfected with plasmids such as TLR4, CD14, MD2 and luciferasefollowing NF-κB promoter. 24 hours after transfection, cells wereincubated with 10 μg/ml purified octameric NID-H protein. The luciferaseactivity is dependent on the transcription level of luciferase gene bytranscriptional factor, phosphorated NF-κB, hence it was used torepresent the activation of NF-κB. The luciferase activity trigged byNID-H was not strong but obvious (FIG. 7a ). The activity of LPS can beblocked by polymyxin B, an effective antibiotic for Gram-negativeinfections. However, the activity of NID-H was not affected. SinceIFP35(ΔN), a N-terminal truncation of IFP35, has high efficiency totrigger the inflammation response, its abilities to active transcriptionfactors were also detected. The results showed that purified ΔN inducedstrong NF-κB and AP-1 promoter activities. In contrast, it has noeffects to IRF3 promoter (FIG. 7b ). These results suggest that IFP35active NF-κB and AP-1 but not IRF-3, probably through Myd88 dependentpathway and the mechanism of IFP35 family members should be parallel tothe known DAMPs. After stimulation by interferon, IFP35 family memberscan be released as danger signal, then lead to cytokine storm throughTLR4-MyD88-NF-κB/AP-1 signaling pathway.

Discussion

In this example, the structure of the NID domain, the characteristicdomain of IFP35 family members, was determined. It is found that theopen conformation of NID-H could activate the innate immune response.The results indicate that the IFP35 family members can act as earlyDAMPs and activate transcriptional factor NF-κB through the TLR4 pathwayand lead to cytokine storm. However, the structures and functions ofIFP35 family members still need be discussed and illustrated.

1. The Structures of the Tandem NID Domains

There are two tandem NID domains in both IFP35 and NMI. The structure ofNID-H crosses two predicted NID domains in IFP35. It could be dividedinto two fragments, β1-α1-β2 (termed as “A part”) and β3-α2-β4-β5(termed as “B part”), covering 131-179 and 180-228 amino acids in thesequence, respectively. Based on the secondary structure predictionresults, the fragment covering 80-130 contains β-α-β-β secondarystructure elements, and it was termed “B′ part”, compared to B part.Similarly, an “A′ part” covering 229-276 contains β-α-β secondarystructure elements. By sequence alignment, parts A′ and B′ arecomparable with parts A and B (FIG. 8a ). Residues involved in thehydrophobic core of NID-H structure were found out as shown in FIG. 8b .These residues are almost completely conserved in these two groups, Awith A′ and B with B′ (FIG. 8a ). Therefore, members in the same groupcan replace with each other. According to previous domain predication, Aand B′ belong to the first NID domain, while A′ and B belong to thesecond. According to the structure of NID-H, the structure of the twotandem NID domains in IFP35 could fold into a double-barrel structure(FIG. 8c ).

In this model, the NID-H region, consisting of part A and part B, existas the open conformation. The skeletons of the tandem NIDs model andNID-H o-dimer structure are similar. Nevertheless, there are manydifferences between them. For example, the surface residues in A′ and B′are not exactly same with those in A and B. Besides, it is reasonablethat the relative position of these two NIDs should be flexible, whichmight be adjusted during receptor recognization. In contrast, theo-dimer conformation of the NID-H is stable because it is fixed in ringstructure of the octamer. Based on these, it is understandable that theNID-H octamer only has about 5-10% activity compared with ΔN. A longloop (LL) is supposed to be existed in the putative NID domain. Itconnects the two parts of the NID domain, which fold into aβ-α-β-β-LL-β-α-β structure. In IFP35, these two long loops cover 115-130and 220-228 amino acids, respectively. The features of these amino acidsindicate that these two loops are flexible. Therefore, these two loopsmight take part in the HMMC formation in cells or receptor recognitionon the out membrane. The structure and functional mechanism of NMI, thehomologous protein of IFP35 could also be inferred from the structure ofIFP35 (FIG. 9).

2. The Recognition Between IFP35 and TLR4

To verify the recognition between IFP35 and TLR4, an in vitro bindingassay was used to pull down IFP35 by TLR4, TLR4/MD2 and TLR4/MD2/CD14complex. The bait was fused with 6*His and immobilized on Ni-NTA beads.The treated beads were used to pull down full-length or truncated IFP35proteins, including purified NID-H octamer, ΔN, and released IFP35 inRAW cell culture. The results showed that only the release IFP35 couldbe captured by TLR4/MD2 complex in the presence of CD14 (Data notshown). Therefore, TLR4, MD2 and CD14 can work coordinately during IFP35recognition.

3. The Differences Between IFP35 Family Members and Known DAMPs

Although the IFP35 family members can stimulate NF-κB through TLR4pathway, similar to the known late DAMPs, in fact, they are expected tohave more differences. As early DAMPs, IFP35 and NMI are released in thefirst hour after cells are stimulated by pathogens, compared to the 6-8hours for HMGB-1. Playing crucial role in the early state of immuneresponse, IFP35 family members may crosstalk with known other earlyinflammatory factors such as TNF-α, IL-1β. From the results herein, theantibody of NID-H are effective to down regulate the expression of theseinflammatory cytokines, so it could provide more efficient protectionfor LPS challenged mice. Besides, their concentrations in patients'serum are far from that of HMGB-1, but comparable to other cytokines,they are more likely to work as signal molecules rather than effectors.

On one hand, IFP35 tends to aggregate because it contains a hydrophobicL-zip domain, on the other hand, it is easy to degrade due to theflexibility of two NID domains. Therefore, IFP35 protein could not foldwell in many expression systems. The released IFP35 by immune cells arelimited and glycosylated. A NID-H fragment which folds into an activeoctameric conformation has a high yield in E. coli. Since the activeconformation is stabilized and the binding surface is exposed in theoctamer structure, the NID-H octamer will be very powerful formonoclonal antibody screening. A monoclonal antibody of NID-Hneutralized IFP35 and provided protection for septic mice. Besides,soluble AN could also be used to prepare monoclonal antibody. Eventhough further evidences are still needed, their applications infighting against infections and injuries could be expected.

These data demonstrated a type of early DAMP, IFP35 family members. Theyare released when the immune cells are stimulated by pathogen orinterferon, and further enhance the immune response through TLR4 Signalpathway. The structure of NID-H shed light on the mechanism of these newDAMPs. The results herein provide a way in diagnosis and treatment forinfection and injury related diseases.

Methods

Plasmid Construction.

The cDNA of Homo sapiens IFP35 and Mus musculus NMI were amplified fromreverse-transcribed cDNA from THP1 and RAW264.7 cells (accession No.:NP_005524.2 and NP_001135421.1). Standard methods for primer design, PCRamplification, digestion and recovery were used. The DNA sequencescorresponding to amino acids 124-240 and 35-289 of human IPF35 protein(NID-H and ΔN) were inserted into pGEX-6p-1 vector (Invitrogen) usingthe BamH I and Xho I sites with an N-terminal GST tag. And the DNAsequences of mouse NMI protein was inserted into RSFDuet vector(Novagen) using the BamH I and Xho I sites. All the plasmids wereverified by DNA sequencing.

IFP35 (NID-H and ΔN) Purification.

The plasmids were transformed into Escherichia coli BL21 (DE3)expression strain (for native protein expression) and Escherichia coliB834 (DE3) expression strain (for Selenomethionine derivative (Se-Met)expression). Cells for native or Se-Met protein expression were culturedin the presence of 0.1 g/L Ampicillin in Lenox Broth medium or inSelenoMet Medium Base (Molecular Dimensions Limited) at 37° C. until theOD₆₀₀ reached 0.8-1.0 and were then induced with 0.5 mMisopropyl-β-D-thiogalactopyranoside (IPTG) at 37° C. for 5 hours.

Cells containing over-expressed native or Se-Met IFP35 (residues124-220) were harvested and resuspended in cold 1×PBS buffer (137 mMNaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4), and lysed bypassing the cell suspension two times through an EmulsiFlex-C5homogenizer (Avestin) at 5,000 psi and 15,000 psi, respectively. Celllysate was centrifuged at 30,700 g/4° C./40 min and the supernatant wasincubated with Glutathione Sepharose 4B resin (GE Healthcare) at a ratioof 100 ml supernatant per ml resin at 4° C. for 30-60 min. Afterincubation, the GST resin was washed with 1×PBS buffer and equilibratedwith a buffer of 150 mM NaCl, 20 mM Tris-HCl, pH 8.0. The recombinantprotein was eluted with 20 mM GSH and the GST tag was cleaved overnightat 4° C. by PreScission Protease (PPase). After cleavage, therecombinant protein was further purified through a HiTrap Q HP (GEHealthcare) column and eluted with a linear gradient of 150 to 1,000 mMNaCl. A HiLoad 16/60 Superdex 200 (GE Healthcare) column (prep grade)equilibrated with 150 mM NaCl, 20 mM Tris-HCl, pH 8.0 was used as thelast purification step. Protein content of each fraction from elutionpeaks was analyzed by SDS-PAGE. Two peaks with target protein werepooled separately, concentrated, flash-frozen in liquid nitrogen, andstored at −80° C. until further use.

NMI, GFP-NID-H and GFP-NMI Purification.

The plasmids were transformed into E. coli strain BL21 (DE3). Cells werecultured in LB medium at 37° C. with 100 mg/L ampicillin. When the OD600reached 0.8-1.0, the culture was induced by addition ofisopropyl-thio-D-galactosidase (IPTG) (Sigma) to a final concentrationof 0.5 mM for 20 h at 16° C. Cells were harvested by centrifugation at5000 rpm for 10 min. Pellets were resuspended in Tris buffer (20 mM Trisat pH 8.0, 150 mM NaCl) and lysed by sonication. The lysate wasseparated by centrifugation at 16,000 rpm for 30 min, and the recoveredsupernatant was applied to a Ni-NTA affinity column (Qiagen), followedby intensive washing with washing buffer (20 mM Tris at pH 8.0, 150 mMNaCl, 50 mM imidazole). Recombinant protein was eluted from the Ni-NTAaffinity column using elution buffer (20 mM Tris at pH 8.0, 150 mM NaCl,500 mM imidazole) and further purified by gel filtration with aSuperdex200 column (GE Healthcare) using Tris buffer as described aboveon an FPLC protein purification system.

The purity and integrity of all recombinant proteins were verified byCoomassie blue staining after SDS-PAGE, with a puritypredominantly >90%. The LPS content in all proteins preparations isundetectable or <10 pg/mg protein as measured by Limulus assay.

Crystallization and Data Collection

Both the native and Se-Met IFP35 fragments exhibited two oligomericstates on the Superdex 200 column: octamer and dimer. Both forms ofprotein were used for crystallization screening. Ultimately, the nativeIFP35 (residues 124-220) was crystallized in its octameric form by thehanging drop method with a drop consisting of 1 μL 6 mg/mL protein and 1μL well solution (0.2 M (NH₄)₂SO₄, 0.1 M Bis-Tris-HCl, 20% [w/v]PEG3350, pH 5.4) at 16° C. for 14 days. Crystals of Se-Met IFP35(residues 124-220) were grown in its dimeric form by the hanging dropmethod from a solution consisting of 1 μL 10 mg/mL protein and 1 μL wellsolution (0.2 M (NH₄)₂SO₄, 0.1 M Bis-Tris-HCl, 22% [w/v] PEG3350, 0.1 MK Na Tartrate, pH 5.5) at 16° C. for 50 days.

Crystals were flash-frozen in liquid nitrogen until data collection. ForSe-Met crystals, an additional 20% [v/v] Glycerol was used as acryo-protectant. The X-ray diffraction data of native and Se-Metproteins were collected on beamline BL17U at Shanghai SynchrotronRadiation Facility (SSRF) at wavelengths of 0.9798 Å and 0.9792 Å,respectively. The collected data were integrated and scaled usingHKL-2000 (Otwinowski, Methods in Enzymology 276, 307-326 (1997)),Selenium-labeled positions in Se-Met protein were determined usingSHELXD from the CCP4 suite (Schneider et al., Acta Crystallographica,Section D, Biological Crystallography 58, 1772-1779 (2002); and Winn etal., Acta Crystallographica, Section D, Biological Crystallography 67,235-242 (2011)) and six selenium sites were found.

Structure Determination and Refinement.

The structure of IFP35 (residues 124-220) in dimeric form was determinedusing SAD in the Phenix suite. Adams et al., Acta Crystallographica,Section D, Biological Crystallography 66, 213-221 (2010). AutoSol wasused to obtain the initial phase. The missing region in the model wasrebuilt with AutoBuild or Coot. Emsley et al., Acta Crystallographica,Section D, Biological Crystallography 66, 486-501 (2010). The model wasfurther refined with phenix.refine in iterative cycles until R valuesconverged. Six molecules were found in one asymmetric unit with aMatthews coefficient value calculated to 2.39 Å³ Da⁻¹ and the solventcontent was 48.6%. Matthews, J Mol Biol 33, 491-497 (1968).

Structure of the octameric form was solved by molecular replacementusing Phaser-MR (Adams et al., Acta Crystallographica, Section D,Biological Crystallography 66, 213-221 (2010)) and the dimeric structureas a search model. The model was rebuilt with Coot (Emsley et al., ActaCrystallographica, Section D, Biological Crystallography 66, 486-501(2010)) and refined with phenix.refine. Adams et al., ActaCrystallographica, Section D, Biological Crystallography 66, 213-221(2010). At the final step, TLS refinement was introduced and the TLSgroups were suggested by the TLSMD server. Painter et al., ActaCrystallographica, Section D, Biological Crystallography 62, 439-450(2006). Eight molecules were found in the asymmetric unit. Thecalculated Matthews coefficient and solvent content were 2.61 Å³ Da⁻¹and 52.8%, respectively. Matthews, J Mol Biol 33, 491-497 (1968).Inter-molecular interactions were analyzed by PISA. Krissinel et al., JMol Biol 372, 774-797 (2007). All crystal structure-related figures wereprepared with PyMOL (www.pymol.org).

Antibodies and Reagents.

Anti-IFP35 antibody was obtained from Abnova (D01P). Anti-HMGB1 antibodywas obtained from Abcam (ab79823). Anti-P3-Actin antibody was obtainedfrom Sigma-Aldrich (A5441). Anti-CD11b (M1/70) and Gr-1 (RB6-8C5)antibody was obtained from BD Biosciences. Anti-IkBa antibody and pIkBaantibody were obtained from Abcam. IFNγ, trichloroacetic acid (TCA),Lipopolysaccharide (LPS) from E. coli 055:5 and D-galactosamine (D-gal)were purchased from Sigma-Aldrich. MCSF was purchased from Peprotech.

Cells and Cell Culture Conditions.

RAW 264.7 macrophages (from ATCC) were cultured in high-glucoseDulbecco's modified Eagle's medium supplemented with 10% fetal bovineserum at 37° C. B6/C57 mice-derived wt, Tlr3^(−/−), Tlr4^(−/−),Tlr7^(−/−), Tlr9^(−/−), Myd88^(−/−) BMDM cells were cultured inhigh-glucose Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum and 20 ng/ml MCSF at 37° C. THP1 cells were culturedin RPMI 1640 medium supplemented with 10% fetal bovine serum at 37° C.RAW 264.7 macrophages, BMDM and THP1 cells were seeded in 6-well platesat a density of 2×10⁶ cells/well and grown overnight. Then the cellswere stimulated with LPS (100 ng/mL), varying concentrations ofSalmonella SR-11, IFN-γ, purified recombinant proteins IFP35-NID or NMIfor different times as indicated in the figure legends.

RNA Isolation and q-PCR.

The total RNA was extracted from RAW 264.7, THP1 and BMDM cells usingTrizol reagent (Invitrogen) according to the manufacturer'sinstructions. The first strand of cDNA was synthesized from 1 ug oftotal RNA using random primers and MMLV reverse transcriptase(Invitrogen). Real-time RT-PCR was performed using the SYBR Green PCRkit (Bio-Rad) and Real-time quantitative polymerase chain reactionanalyses were performed using the CFX96 Real-Time PCR System (Bio-Rad).Each measurement was set up in duplicates, and three independentexperiments were performed. The primer sequences were as follows:

mouse (m) GAPDH: sense, CAGAACATCATCCCTGCATC; antisense,TACTTGGCAGGTTTCTCCAG; mTNFα: sense, CCAGTGTGGGAAGCTGTCTT; antisense,AAGCAAAAGAGGAGGCAACA; mIL-1β: sense, AAGGAGAACCAAGCAACGACAAAA;antisense, TGGGGAACTCTGCAGACTCAAACT.

Western Blot.

RAW 264.7 macrophages and THP1 cells were pretreated with 100 ng/mL LPSor Salmonella SR-11 for 1 h, 2 h, 3 h, 5 h and 9 h. Then the cells werewashed twice with cold PBS, scraped, and collected in lysis buffer (20mMTris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 10% glycerol,protease inhibitor cocktail (Roche)). The whole cell lysates wereincubated at 4° C. for 45 min, followed by centrifugation (12 000 g×15min, 4° C.). Secretory protein in the cell culture supernatant wascollected by trichloroacetic acid (TCA)/acetone precipitation. The cellculture supernatant was added 0.11 volumes of ice-cold 100% TCA andplaced on ice for 2 h, followed by centrifuge at 20,000 g for 30 min.Then carefully remove the supernatant, add 500 μL of acetone, centrifugeat 20,000 g for 10 min, carefully remove the supernatant and dry theprotein pellet in a vacuum evaporator. Protein samples were separated bySDS-PAGE, transferred to a PVDF membrane, and probed with specificantibodies against IFP35, HMGB1, and NMI.

BMDM cells were treated with purified recombinant proteins IFP35-NID orNMI for 5 min, 15 min, 30 min, 60 min. Then the cells were washed twicewith cold PBS, scraped, and collected in lysis buffer. The whole celllysates were incubated at 4° C. for 45 min, followed by centrifugation.Protein samples were prepared for western blot. IkBa, pIkBa, β-Actinwere detected by WB.

LPS-Induced Shock Model.

LPS from E. coli 055:B5 and D-gal were diluted in pyrogen-free saline. Acombination of LPS (50 μg per kg body weight) and D-gal (1.0 g per kgbody weight) was injected intraperitoneally in IFP35^(−/−), NMI^(−/−)and wild-type mice. Resistant of IFP35^(−/−) and NMI^(−/−) mice to LPSinduced lethl toxicity were also observed when mice were injected with alarge amount of LPS (50 mg per kg body weight) without D-gal. Observeand record the mortality of mice for 1 week.

IFP35 Monoclonal Antibody Protects LPS Shocked Mice.

In the survival experiment, IFP35 mAb and IgG1 (10 μg per mouse) wereinjected intraperitoneally in B6/C57 mice 4 h before the injection ofLPS (50 mg per kg body weight). Additional doses (10 μg per mouse) wereadministered at 2 h after LPS injection. Delayed doses (10 μg per mouse)was administrated every 24 hours for 4 times. Observe and record themortality of mice for week.

Determination of Cytokine Concentrations.

IFP35 and NMI in human and mouse serum were measured with ELISA kits(CUSABIO). Release of cytokines (IL-1β, IL-6, and TNFα) in the culturesupernatants or in mouse serum were measured by ELISA Kit (Biolegend,Thermo Fisher).

Generation of IFP35^(−/−) Mice.

Genomic engineering of IFP35 was achieved with the CRISPR/Cas9 system asdescribed.

Flow Cytometry.

Recombinant protein IFP35 NID-H, NMI or PBS were injectedintraperitoneally in B6/C57 mice for 24 h, respectively. The mouseascites was incubated with antibody to CD11b and Gr-1, followed byFITC-conjugated secondary antibody. Cells were analyzed on a FACSCaliburmachine (BD Biosciences) to detect the peritoneal neutrophils and datawere analyzed using CellQuestPro software.

B6/C57 mice-derived wt or Tlr4^(−/−) cells were incubated with GFP-NID-Hand GFP-NMI in PBS buffer (containing 2% FBS) for 1 h. Cells wereanalyzed on a FACSCalibur machine (BD Biosciences) and data wereanalyzed using CellQuestPro software.

Citation of the publications or documents herein is not intended as anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents.

Example 2: Use of IFP35 Family Proteins

IFP35 and NMI can be rapidly up-regulated by interferon (IFN). Theprotein level of IFP35 in cells is low without the induction of IFN.IFP35 and NMI can be induced by type I or type II interferon (IFNα/β/γ)in numerous immune cells. The amount of IFP35 protein and mRNA levelincreased dramatically after stimulation of IFN γ for 6 h and reachedthe maximum level at 24 h, revealed a 25-fold increase compared withnon-treatment. Most cells can express NMI, and similarity with IFP35,the expression of NMI increased 2-20 fold after IFN treatment.

Homo sapiens IFP35 is located in the 17q21, with a NCBI Accession No.NP_005524.2. The cDNA sequence encodes a 288-amino acid protein with adeduced molecular mass of 31.8 KDa. A natural mutant with methionine atposition 128 mutated and/or modified to valine (M128V) exists. Homosapiens NMI is located on chromosome 2, with a NCBI Accession No.AAC12949.1, including 307 amino acids.

IFP35 and NMI are homologous protein. According to the structureprediction, they both include two tandem N-Myc binding protein/IFP35domain (Nmi/IFP35 domains, NIDs). According to sequence analysis, IFP35has an L-zip domain at its N-terminus containing the first 80 aminoacids, following by two tandem NID domains covering the 81-170 and177-268 amino acids, respectively.

IFP35 and NMI are co-located in the cytoplasm in different cell types byimmunofluorescence microscopy techniques. NMI and IFP35 proteins canform a high molecular mass complex (HMMC) of 300-400 kDa through NIDdomain as determined by native gel electrophoresis and gel filtrationwhich suppress the IFP35 degradation by proteasome. IFP35 and NMI canform speckle-like aggregations after stimulation of IFNγ calledNMI/IFP35 speckles (NIS). In IFNγ-treated apoptotic cells, the NISdissociates, but the high molecular mass complex does not dissociate.

It is reported that IFP35 can regard as antiviral protein and inhibitthe Bovine Foamy Virus replication. The second NID domain of IFP35 canbind the long terminal repeat sequences of early regulatory protein BTas(Bovine transactivator protein) of Bovine Foamy Virus, thus inhibitingthe transcription activity to suppress the virus infecting cells. Inaddition, high throughput screening showed that IFP35 protein couldinteract with CLEC4G (C-type lectin domain family member 4 G) in theRas-MAPK/PI3K pathway.

NMI can participate in the JAK-STAT pathway induced by IFN. NMIinteracts with all STATs except Stat2, and enhances the association ofmutual activation factor CBP/p300, thus enhances STATs-mediatedtranscription level of downstream genes.

The mRNA level of NMI in malignant breast cancer cell lines decreased25-45 fold compared to the normal breast cells and the protein level ofNMI was also significantly lower than the normal breast cells.Continuous telomerase activity is considered as one of the prerequisitefor cancer, and NMI can bind breast cancer susceptibility protein BRCA1(breast cancer type 1 susceptibility protein) and c-Myc to form aternary complexes and down-regulate the promoter activity ofanthropogenic telomerase reverse transcriptase gene (human telomerasereverse transcriptase gene, hTERT). hTERT promoter activity wasdecreased by about 75% when co-transfected the Flag-Nmi and HA-BRCA1into cells. In addition, when detected the protein Dkk1 (Dickkopf-1) inthe Wnt/β-catenin pathway, the data suggest that overexpression NMIinhibits the Wnt/β-catenin signaling via up-regulation of Dkk1 andretards tumor growth.

NMI can up-regulate the negative feedback regulating factors SMAD7 inthe TGF/SMAD β signaling pathway to inhibit TGF/SMAD β signalingpathways and reduce the aggressiveness and mobility of the breast cancercells. IFP35 and NMI are associated with inflammatory signals.

FIG. 10 illustrates the process of IFP35 protein purification. (A)SDS-PAGE analysis of IFP35-NID. The left lane: molecule marker. Lane 1:GST-tagged IFP35-NID after purification of affinity chromatography. Lane2: the mixture of proteins after digestion by PPase. Lane 3: theIFP35-NID eluted from the GST resin column. Lane 4: IFP35-NID purifiedafter size-exclusion chromatography. (B) Size-exclusion chromatographyanalysis (superdex-200(16/60)). The elution volume of the peaksuggesting the aggression state of the protein is close to dimer

FIG. 11 shows the different oligomer states of IFP35. (A) Anion exchangecolumn analysis. (B) Size-exclusion chromatography analysis. The elutionvolumes of two peaks suggesting different oligomer formation. (C)SDS-PAGE analysis for the whole process of purification. Lane 1:GST-tagged IFP35. Lane 2: IFP35 eluted from the GST column. Lane 3:IFP35 after digestion by PPase. Lane 4: sample binding to anion exchangecolumn. Lane 5: sample that did not bind to anion exchange column. Lane6: protein eluted from size-exclusion chromatography superdex-200 (theelution volume of the peak was 72-74 ml). Lane 7: protein eluted fromsize-exclusion chromatography superdex-200 (the elution volume of thepeak was 90-92 ml).

FIG. 12 shows the IFP35-NID crystal. (A) Native octamer crystal. Theprotein was crystallized in reservoir solution with 0.2 M ammoniumsulfate, 0.1M Bis-tris, 25% [w/v] PEG3350, pH5.5, in which the drop wasset up at 1 μl protein and 1 μl reservoir solution. The crystal wasgrown for 14 days at the temperature of 289 k. (B) Native octamercrystal. The native IFP35-NID (residues 124-220) was crystallized in itsoctameric form by the hanging drop method with a drop containing 1 μLprotein and 1 μL well solution (0.2 M (NH4)₂SO₄, 0.1 M Bis-Tris-HCl, 20%[w/v] PEG3350, pH 5.4), with 30% [w/v] d-glucose as an additive, at 16°C. for 1-14 days. (C) Crystals of Se-Met IFP35-NID (residues 124-220)were grown in its dimeric form, with the hanging drop method from asolution consisting of 1 μL protein and 1 μL well solution (0.2 M(NH4)₂SO₄, 0.1 M Bis-Tris-HCl, 22% [w/v] PEG3350, pH 5.4) at 16° C. for7 days. Scale bar=100 μm. (D) Crystals of Se-Met IFP35 (residues124-220) were grown in its dimeric form, with the hanging drop methodfrom a solution consisting of 1 μL protein and 1 μL well solution (0.2 M(NH4)₂SO₄, 0.1 M Bis-Tris-HCl, 22% [w/v] PEG3350, pH 5.5) at 16° C. for30 days. Scale bar=200 μm. The crystals are identified with arrows.

FIG. 13 shows the structure of the dimeric IFP35-NID. (A) and (B) arethe whole structure from different views. (C) and (D) indicate residues(shown in sticks) involved in the intra-molecule interaction fromdifferent views. H1 indicates α-helix mainly involved in dimerformation. A single molecule is shown in red or blue.

FIG. 14 shows the structure of the octameric IFP35-NID. Monomers areshown in different colors. The octamer were formed with fourdomain-swapping dimers in the manner similar to the interaction thatform the dimer. The amino terminal and carboxyl terminal of molecule Aare indicated with A-N and A-C.

FIG. 15 shows the structure of dimer with domain-swapping conformation.The domain-swapping model of octamer. A-dimer and B-dimer are extractedfrom the octamer. Red and blue indicate one molecule respectively. Twomolecules can bind to each other. The dimers finally form a ringstructure of octamer through interaction between the side H1 α-helix ofone dimer and the H1α-helix of its neighbor dimer.

FIG. 16 shows the residues of some areas. (A) Residues on the arcintersecting surface area (atoms on side chain are labelled and shown insticks, atoms on the main chain are not labelled). (B) Residues on theinner surface of the octamer ring structure (atoms on side chain arelabelled and shown in sticks, atoms on the main chain are not labelled).(C) Residues on the head of a monomer within a dimer (from twomolecules, atoms on side chain are labelled and shown in sticks, atomson the main chain are not labelled). The whole structure are shown inribbons, different colors indicate different molecules.

FIG. 17 shows the purification of the recombinant NMI. (A) The SDS-PAGEof the mouse NMI-NID fragment. (B) The size exclusion chromatographyresults of NMI-NID. (C) The SDS-PAGE of the full length mouse NMI with6*His tag. (D) The size exclusion chromatography result of full lengthmouse NMI with 6*His tag at its N terminal.

FIG. 18 reveals the expression level of IFP35 and HMGB1 (as a control)when stimulated by salmonella.

FIG. 19 shows that the abundance of IFP35 changed with time as measuredby immunofluorescence when stimulated by salmonella.

FIG. 20 shows the abundance of IFP35 secreted to the medium at differentperiods when cells are infected by the virus. (A) The abundance ofsecreted IFP35 at different times measured by western blot while thecells are infected by A59 and MHV68 with different MOI. (B) Theabundance of secreted HMGB1 at different times measured by western blotwhile the cells are infected by A59 and MHV68 with different MOI.

FIG. 21 shows a model of mouse peritonitis. (A) In the model ofperitonitis induced by mouse Standard strains of salmonella typhimuriumSR-11, IFP35 was released to the medium when injected live SR-11 to themodel. (B) IFP35 are released to the medium in case of that inflammasomeis stimulated by LPS and ATP.

FIG. 22 shows the contribution of the IFP35 with different oligomerstates during the process of stimulating inflammation. Expression levelof some inflammation factors are measured as indicated in the figure.

FIG. 23 shows the result of Flow cytometry analysis, which illustratesoctameric IFP35-nid can recruit large amount of neutrophil granulocytes.

FIG. 24 shows exogenous octameric IFP35-NID can stimulate NF-κB pathwayin macrophages.

FIG. 25 illustrates that IFP35 can stimulate inflammation through myd88signal pathway.

FIG. 26 shows the levels of IL-1β and TNF among WT, Tlr9^(−/−) andTlr4^(−/−) mouse as induced by IFP35.

FIG. 27A shows the amount of IFP35 in the serum of septic mice. FIG. 27Bshows the survival rate of the septic mice was increased whenadministrated with IFP35 monoclonal antibodies.

FIG. 28 shows that the block of IFP35 attenuated the release of inducedIL-6 by LPS in mice.

FIG. 29 shows the abundance of NMI in cell lysate and supernatant ofThp1 cells stimulated by salmonella.

FIG. 30 demonstrates that mouse source NMI protein can up-regulate thetranscription of TNFα and IL-1β in Thp1 cells.

FIG. 31 shows the SDS-PAGE detection of the aggregation state of NMI byrunning size exclusion chromatography. The digits labeled on eachprotein lanes indicate elution volume. Since there is a sustained NMIelution profile, the result suggested that NMI may exist as differentoligomers in mouse serum.

FIG. 32 shows a secondary structure prediction of human IFP35 (performedby PSIPred (http://bioinf.cs.ucl.ac.uk/psipred/)).

The experimental methods used in the following protocols are allconventional methods unless otherwise specified. All the materials andreagents used in the following protocols can be commercially obtainedunless otherwise specified. The results of IFP35-NID1 and IFP35-NID2 inthe following protocols have no significant differences.

The expression, purification, and crystallization of IFP35 and NMI.

1. Plasmid Construction

The cDNA of Homo sapiens IFP35 and Mus musculus NMI were amplified fromreverse-transcribed cDNA from THP1 and RAW264.7 cells (Accession No.:NP_005524.2 and NP_001135421.1). The sequence of IFP35 was verified tobe the wild variant of M128V by DNA sequencing.

SEQ ID NO: 1 is the cDNA sequence of Homo sapiens IFP35. SEQ ID NO: 2 isthe amino acid sequence of Homo sapiens IFP35. In one aspect, a NID ofIFP35 is encoded by nucleic acid 372 to 660 of SEQ ID NO: 1 (from the 5′end), which encodes amino acid residues 124 to 220 of SEQ ID NO: 2 (fromthe N-terminus). In one aspect, a NID of IFP35 is encoded by nucleicacid 408 to 648 of SEQ ID NO: 1 (from the 5′ end), which encodes aminoacid residues 136 to 216 of SEQ ID NO: 2 (from the N-terminus).

SEQ ID NO: 3 is the cDNA sequence of Mus musculus IFP35. SEQ ID NO: 4 isthe amino acid sequence of Mus musculus IFP35.

SEQ ID NO: 5 is the cDNA sequence of Mus musculus NMI. SEQ ID NO: 6 isthe amino acid sequence of Mus musculus NMI. NMI-NID is encoded bynucleic acid 453 to 750 of SEQ ID NO: 5 (from the 5′ end), which encodesamino acid residues 151 to 250 of SEQ ID NO: 6 (from the N-terminus).

SEQ ID NO: 7 is the cDNA sequence of Homo sapiens NMI. SEQ ID NO: 8 isthe amino acid sequence of Homo sapiens NMI. NMI-NID is encoded bynucleic acid 465 to 720 of SEQ ID NO: 7 (from the 5′ end), which encodesamino acid residues 155 to 240 of SEQ ID NO: 8 (from the N-terminus).

The PCR primers used in this protocol are synthesized by BeijingSynthesis, Sangon Biological Engineering (Shanghai) Co., LTD. Thesuperstar high-fidelity DNA polymerases used were purchased from GenStarBiosolutions Co., Ltd. The restriction endonucleases were bought fromTakara biotechnology (Dalian) Co., LTD. The agarose was purchased fromBiowest (US). The quick gel extraction kit was purchased from BeijingTransgen Biotechnology Co., LTD. The E. coli expression vector pGEX-6p-1is from GE Healthcare Incorporation. The DH5α, BL21 (DE3) chemicallycompetent cells and B834 defect competent cells are purchased fromInvitrogen Company. DNA sequencing was performed by Beijing LiuheGenomics Technology Co., LTD.

2. Protein Expression and Purification

Tryptone and yeast extracts used to cultivate E. coli are purchased fromThermo Fisher Oxoid (UK). High pressure homogenization cell crackerEmulsiFlex-C5 used for cell crash is purchased from Avestin (Canada).Prescission protease (Ppase) is purified from a E. coli strain. GSTaffinity column (Glutathione Sepharose 4B), anion exchange column(HiTrap_Q_HP_5 ml), size-exclusion chromatography column(HiLoad_16/60_Superdex200_prep grad and Superdex 200 10/300 GL, 24 ml),protein purification system (ÄKTApurifier) are purchased from GEhealthcare life sciences. The NanoDrop 2000 spectrophotometer used forconcentration detection is purchased from Thermo (US).

Other biochemical Reagents used are purchased from Sigma Aldrich Companyor Beijing Chemical Reagent Company.

3. Crystallization and Data Collection

Crystallization screening kits and Se-Met are purchased from HamptonResearch Incorporation. Buffers including Tris-base, Bis-Tris, andpolymer PEG3350 are purchased from Sigma-Aldrich (US). Home Source X-raydiffractometer is purchased from Rigaku Incorporation (Japan).

IFP35 Expression, Purification, and Crystallization

For purification, many tags such as 6×HIS, GST, or MBP can be chosen.Take GST-tagged protein as an example, the DNA sequences correspondingto amino acids 1-288, 128-216 and 136-216 of Homo sapiens IPF35 proteinwere inserted into pGEX-6p-1 vector (Invitrogen) using the BamH I andXho I sites with an N-terminal GST tag. All the plasmids were verifiedby DNA sequencing. The recombinant protein obtained in this way can becleaved by PreScission Protease (PPase). The recombinant plasmids offull-length IFP35 and other fragments are constructed in the similarway.

pGEX-6p-1 vector has GST tag and PPase cleavage site in front of themultiple cloning site.

(1). Plasmid Construction

Take the cDNA sequence of IFP35 as the template, pairs of primers forPCR amplification were used to get full length IFP35 protein-codinggene, IFP35 NID1 encoding gene, IFP35 NID2 encoding gene respectively,which were digested by BamH1 and EcoRI restriction endonucleases andthen ligated to the vector pGEX-6p-1 cleaved by the same restrictionenzymes. Following the ligation reaction, the ligated plasmid DNA wastransformed into competent cells such as E. coli DH5a (Invitrogen) usingheat-shock method. The bacteria were spread thin on the plate withconsistent antibiotic resistance (100 μg/ml ampicillin) and plates wereincubated at 37° C. overnight. The next day, several colonies werepicked into 2 ml LB medium and cultivated at 37° C. for 10 h for plasmidextraction to confirm the presence of the recombinant plasmid by DNAsequencing. Once it has been established that the insert is in theproper orientation and the correct junctions are present, the plasmidsare used for protein expression.

By DNA sequencing, Plasmid 1 is the GST-IFP35 recombinant plasmidcontaining SEQ ID NO: 1 inserted into pGEX-6p-1 using the BamH I and XhoI sites. Plasmid 2 is the GST-IFP35-NID1 recombinant plasmid containingDNA sequence 372-660 from the 5′ end of SEQ ID NO: 1, which is insertedinto pGEX-6p-1 using the BamH I and Xho I sites and was added with atermination codon. Plasmid 3 is the GST-IFP35-NID2 recombinant plasmidcontaining DNA sequence 408-648 from the 5′ end of SEQ ID NO: 1, whichis inserted into pGEX-6p-1 using the BamH I and Xho I sites and wasadded with a termination codon.

The primers for amplification of full-length IFP35:

Upstream primers: 5′GGTAGATCTATGTCAGCCCCACTGGATG3′ Downstream primers:5′GGT GAATTCCTAGCCTGACTCAGAGGTGAAGACT3′The primers for amplification of IFP35-NID1 (amino acids 124-220 of SEQID NO: 2):

Upstream primers: 5′ GGTGGATCCCCAGGTGATGATGTCCAGCCA3′Downstream primers: 5′GGTGAATTCTTACTCCCCGTTCACATACGGAGAGAC3′The primers for amplification of IFP35-NID2 (amino acids 136-216 of SEQID NO: 2):

Upstream primers: 5′ GGTGAATTCAGGGTGTTGGTCACTGGATTTCCT3′Downstream primers: 5′GGTGAATTCTTACTCCCCGTTCACATACGGAGAGAC3′

(2). Protein Expression

E. coli BL21 (DE3) cells were transformed with the recombinant plasmidsand grown on LB agar plates containing 100 μg/ml ampicillin. Followingovernight incubation at 37° C., single colonies were transferred into100 ml LB broth containing 100 g/ml ampicillin and grown overnight at37° C. with shaking (200 rev min-1). The cells were diluted 1/400 inflasks containing 1 L LB broth supplemented with 100 g ml-1 ampicillinand cultivated at 37° C. with continuous shaking (200 rev min-1) untilan OD600 nm of 0.6 was reached. The cells were subsequently treated with1 mM IPTG to induce the expression of recombinant proteins. After 5 h ofincubation with continuous shaking (200 rev min−1) at 37° C., the cellswere harvested by centrifugation at 4000 rev min−1 for 30 min at 4° C.

Cells containing over-expressed proteins were harvested and resuspended10/1 in cold 1×PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8mM KH2PO4, pH 7.4), and lysed by passing the cell suspension two timesthrough an EmulsiFlex-C5 homogenizer (Avestin) at 5,000 psi and 15,000psi, respectively. Cell lysate was centrifuged at 30,700 g/4° C./40 minand collect the supernatant.

(3). Protein Purification

1) Affinity Chromatography

After centrifugation, the supernatant was incubated with GlutathioneSepharose 4B resin (GE Healthcare) at a ratio of 100 ml supernatant perml resin at 4° C. for 30-60 min. After incubation, the GST resin wasadded to the gravity column for the separation of the resin from thesolution mixture, then the column was washed about 10 resin volumes with1×PBS buffer to bind the GST-tagged proteins on the resin.

Next, the GST resin was equilibrated with a buffer of 150 mM NaCl, 20 mMTris-HCl, pH 8.0. The recombinant protein was cleaved overnight at 4° C.by PreScission Protease (PPase) and then eluted with 2 column volumesbuffer containing 15 mM GSH, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0. at thespeed of 1 mL/min for 30 min. After cleavage and elution, IFP35, IFP35NID1, and IFP35 NID2 (all the elution volumes are about 50 ml) wereobtained.

2) Anion-Exchange Chromatography

Proteins digested by protease PPase enzyme were injected into anionexchange column (HiTrap_Q_HP_5 ml, GE Healthcare company), which isfixed to the FPLC purifier (GE Healthcare) and was balanced with 20mMTris-HCl (PH8.0), 50 mM NaCl before injection. With the elution buffer(20 mMTris HCl (PH8.0), 1 M NaCl) to carry on the gradient elution atthe flow rate of 1 ml/min and the elution volume 100 ml, samples of thecorresponding protein were collected according to the 280 nm and 260 nmultraviolet absorption.

3) Size-Exclusion Chromatography

A HiLoad 16/60 Superdex 200 (GE Healthcare) column (prep grade)equilibrated with 150 mM NaCl, 20 mM Tris-HCl, 5% gly, pH 8.0 was usedas the next purification step after anion-exchange chromatography. Thecolumn was fixed to the FPLC purifier (GE Healthcare) as the same andthe flow rate was 1 ml/min eluted for 120 min. The full-length humanIFP35 protein and IFP35-NID were collected at elution volume of about 45ml and 90-92 ml respectively. Finally, the purified IFP35 (elutionvolume of 45 ml), IFP35 NID1 (elution volume is 90-92 ml), and IFP35NID2 (elution volume is 90-92 ml) were obtained.

Size-exclusion chromatography of IFP35-NID1 and IFP35-NID2 are shown inFIG. 10. The elution volumes of IFP35-NID1 and IFP35-NID2 samples are90-92 ml corresponding to the molecular weight 17-43 KD. Results ofcentrifugal analysis and crystal structure indicated that the IFP35-NID1and IFP35-NID2 were dimers.

During the purification of IFP35-NID1 and IFP35-NID2, two differentoligomer states of the proteins corresponding to the elution volumes of72 ml and 90-92 ml can be obtained (FIG. 11). When the proteins wereeluted down from the resin column before the digestion of PPase, twopeaks, one at volume 72 ml and the other at volume 90-92 ml, wereobserved. The peak at volume 72 ml corresponded to molecular weight of70 kd-100 kd, and the peak at volume 90-92 ml corresponded to the dimer.The peaks of elution volumes of IFP35-NID1 and IFP35-NID2 suggesteddimer and octamer.

The SDS-PAGE analysis of IFP35-NID1 and IFP35-NID2 had no significantdifferences.

4) Se-Met IFP35-NID Purification

The purification of Se-Met IFP35-NID was similar to native IFP35-NID.The difference is the application of Escherichia coli B834 (DE3)expression strain (for Selenomethionine derivative (Se-Met) expression)and the M9 medium with additive Se-Met. Methionine of proteins expressedby b834 with M9 medium was replaced with Se-Met. Crystal obtained inthis method can be used to determine crystal phase and further forstructure determination.

(4). Protein Crystallization

1) Crystallization

Both the native and Se-Met IFP35-nid1 and IFP35-nid2 fragments exhibitedtwo oligomeric states on the Superdex 200 column: octamer and dimer.Both forms of protein were used for crystallization screening performedusing multiple conditions from the commercially available kits (HamptonResearch). The purified proteins are centrifuged at 14000 rpm/10 min/4°C. to remove precipitation and bubble. Crystallization trials werecarried out by the sitting-drop vapor-diffusion method, mixing 1 μl ofthe protein sample with an equal volume of screening solution on theSiliconized slides and equilibrated over 200 μl of the latter in thereservoir. The crystals are grown at 16° C. Crystals of IFP35-NID1 areshown in FIG. 12.

2) Structure Determination and Refinement

In the crystal structure of dimeric IFP35-NID1 or IFP35-NID2 (FIG. 13),there are six molecules within each asymmetric unit, and the structureof the six IFP35-NID1 or IFP35-NID2 molecules are the same. Electrondensity chart can clearly show that residues of amino acids 136 to 216in IFP35, but the crystal structure can not clearly completely displaythe residues (124-135) at the amino terminus and the residues (216-220)at the carboxyl terminus. This phenomenon illustrated that the core ofthe IFP35-NID domain structure is mainly composed of amino acids136-216, the increase or decrease of individual residues at the carboxylterminal and amino terminal is not important to the overall structure.Hence, these residues are dispensable in crystal structure analysis andcrystallization experiments. In IFP35-NID structure, five antiparallelbeta piece (named B1-B5 according to the sequence order respectively)made up the barrel structure, which surrounded an alpha helix (H1) toform a complete barrel structure (FIG. 13A and FIG. 13B).

IFP35-NID2 formed dimer on the interface where alpha helix (H1) locatedthrough hydrogen bonds and hydrophobic interaction, van der Waals forceand so on (FIG. 13C and FIG. 13D). The size of the interaction surfacecalculated by PISA (Protein Interface, Surface and Assemblies) is about586.5 A square. Residues mainly participated in dimer formation includedGlu150, Glu151, Asp155, Lys158, Ile159, Arg165 and Asp172 on the alphahelix (H1).

Eight NID-1 or 2 molecules interacted with each other and formed a ringlike structure. The monomer in octamer is quite similar to the monomerin the dimer that the monomer folds into barrel-like structure formed byfive β-strands and two α-helixes. Several pairs of residues on helix α1in octameric structure play important roles for interaction betweenmolecules and to mediate dimer formation, which are the same as thedimeric structure. As for the difference, the antiparallel β2 and β3(177Lue-216Tyr) in dimer is straightened in octamer and inserted intothe structure of the adjacent molecule being part of the neighbor NIDdomain. In this way, two monomers piled up a dimer and formed adomain-swapping conformation between two monomers, which makes itdifferent from the dimer.

For clarity, this domain-swapping dimer was named open conformationaldimer (o-dimer). By comparison, the above mentioned dimer withoutdomain-swapping is called closed conformational dimer (c-dimer). Theo-dimer folded in the same way as c-dimer, so the structure of theswapping NID domain is similar to that of the monomer in c-dimer (FIG.15). When the adjacent o-dimer interact using the same H1α helix used inc-dimer, the four o-dimer formed the four-angle star structure (FIG.14). The eight monomer was marked A to G started from N-terminal in theclockwise orientation and the whole structure could be divided into fourpairs of o-dimer A/B, C/D, E/F and G/H respectively, among which thestructural details of A/B and E/F or C/D and G/H are similar andstructural differences of the loop distinguished A/B and E/F from C/Dand G/H. For the four domain-swapping dimers (o-dimer) formed thefour-angle star, the diameter of the inner hole is 35 Å and The outerdiameter is 80 Å. The octamer formed a new arc intersecting surfacethrough cross-over of two swapping molecules, which is absent fromc-dimer. The octameric ring structure was absent from c-dimer. Thestructural differences may be the basis for the functional differencesbetween different oligomer states.

Based on the octameric structure, some residues are observed to beinvolved in structure formation, and others are observed to be mainlyexposed on the surface of the whole structure and are supposed to beinteracted with other proteins. These external residues mainlydistributed in three areas as follows:

The domain-swapping dimer form an arc intersecting surface area on whichthe dimer provide some residues such as Ser145, Asp172, Val173, Leu177,Arg212, Gln199, Gln207, Gln208, Pro210, Ser214, Thr201, and Tyr216 toform a large exposed surface (FIG. 16A). Among them, residues Glu150,Glu175, Leu177, Gln207, and Gln208 are in a more prominent position(FIG. 16A).

Other residues on the inner surface of the ring structure formed by theoctameric IFP5-NID can participate in the interaction with otherproteins. These residues include Ser145, Arg147, Glu150, Glu151, Val173,Gly206, Gln207, and Gln208. In some aspects, Arg147, Gln207, Gln208,Glu150, and Glu151 provide extended side chains that protrude from theinner surface of octamer ring structure (FIG. 16B).

Based on the structure of the dimer or the octamer, there are somerelatively large amino acid residues near the top of the amino terminusand the carboxyl terminus of the β sheet barrel-like structure of thesingle monomer or domain-swapping monomer; these relatively large aminoacid residues extend outwards. These residues can include Arg187,Glu188, Gln192, Gln196, Arg212, and Tyr216, and they are close indistance. Furthermore, some amino acid residues may participate inbinding to other proteins, antibodies, or small molecules (FIG. 16C).

3) Data Collection and Structure Determination

The prepared crystal was tested on Rigaku-007 X-ray diffractometer andthe diffraction data were collected on beamline BL17U at ShanghaiSynchrotron Radiation Facility (SSRF). X-ray diffraction data werecalculated with existing common structure determination or analysissoftware or package including data analysis software such as HKL2000 andMosflm, Image processing software coot and pymol, Heavy atoms solvesoftware shelx, and Structure refinement software Phenix and CCP4.Mainly used methods here are Single wavelength anomalous scattering andmolecular displacement.

The X-ray diffraction data of Se-Met proteins were collected on beamlineBL17U at Shanghai Synchrotron Radiation Facility (SSRF) at wavelengthsof 0.979 Å. 720 images were collected with Rotation range lo per imageand were integrated and scaled using HKL-2000. After calculation, theresolution ratio was 2.3 Å and the space group was H3. Selenium-labeledpositions Selenium-labeled positions in Se-Met protein were determinedusing SAD OF SHELXD and six selenium sites were found. AutoSol was usedto obtain the initial phase. The model was rebuilt with AutoBuild andwas further refined with coot and phenix.refine.

The 2.5 Å X-ray diffraction data of native octameric crystal wasobtained at Shanghai Synchrotron Radiation Facility as the same.Structure of the octameric form was solved by molecular replacementusing Phaser-MR in Phenix and the dimeric structure as a search model,and was further refined with coot and phenix.refine.

All the results above indicated that the full-length IFP35 or the NIDfragments predicted could not express or were expressed as high polymer,making it difficult to get proteins with uniform states. However, thesecond half of the first NID domain and the first half of the second NIDdomain (residues 124-220 or 134-216, which are named IFP35-NID1 andIFP35-NID2, respectively, which are collectively referred to asIFP35-NID because of the similar purification results) were expressedand purified well. Using methods above, a large amount of high puritysoluble IFP35-NID can be obtained, especially when they were fused toGST. GST fused protein is referred to as GST-IFP35-NID.

During purification, there were two stable aggregation states ofIFP35-NID and well-diffracted crystals with both states can be obtained.

Expression and Purification of NMI

Take the cDNA sequence of NMI as the template, pairs of primers for PCRamplification were used to get full length human NMI protein-codinggenes (1-924 from 5′ end of SEQ ID NO: 7), human NMI-NID (residuescorresponding to amino acid residues 155-240) encoding gene (465-720from 5′ end of SEQ ID NO: 7), mus musculus NMI encoding gene (1-945 from5′ end of SEQ ID NO: 5), mus musculus NMI-NID (residues corresponding toamino acid residues 151-250) encoding gene (453-750 from 5′ end of SEQID NO: 5), respectively, which were digested by BamH1 and XhoIrestriction endonucleases and then ligated to the vector pGEX-6p-1cleaved by the same restriction enzymes. In this way, the recombinantplasmids that express GST-human-NMI, GST-human-NMI-NID, GST-mouse-NMI,and GST-mouse-NMI-NID, with added termination codon at the 3′ terminus,were obtained.

Primers for amplification of full-length human NMI:

Upstream primer: 5′GGT GGATCC ATGGAAGCTGATAAAGATGAC3′ Downstream primer:5′GGT CTCGAG CTATTCTTCAAAGTATGCTATGTG3′

Primers for amplification of human NMI (155-240):

Upstream primer: 5′GGT GGATCC TCTAAAATGAAAATCAATGTTAC 3′Downstream primer: 5′GGT CTCGAG TTATTCTGTGTATGGAGAAACAG 3

Primers for amplification of full-length mouse NMI:

Upstream primer: 5′CGCGGATCCATGGATGCTGATAAAGACAAC3′ Downstream primer:5′CCGCTCGAGTCATATGGTTTCTCTGGCCTC3′

Primers for amplification of mouse NMI (151-250):

Upstream primer: 5′CGCGGATCCGTTCATGTGGACATTTCTAAAATG3′Downstream primer: 5′CCGCTCGAGTCAAAACACCTGGTACTTTTCTAAG3′

The mouse and human full length or fragments of NMI were expressed usingE. coli prokaryotic expression system. Reagents and expression methodsused are basically the same as IFP35 protein expression.

The protocol for NMI expression was essentially the same with IFP35. Theprotocol of NMI purification was essentially the same with IFP35. Thepurification was divided into 4 steps: GST gravity column affinitychromatography, cleavage of GST with PPase, anion exchangechromatography, and gel filtration chromatography.

1) GST Affinity Chromatography

After centrifugation, the supernatant was incubated with GlutathioneSepharose 4B resin (GE Healthcare) at a ratio of 100 ml supernatant perml resin at 4° C. for 1-2 h. After incubation, the GST resin was addedto the gravity column for the separation of the resin from the solutionmixture. Then the column was washed with 1×PBS buffer until there wereno miscellaneous proteins on the resin.

The GST tagged NMI was eluted the elution buffer containing 10 mM GSHand then cleaved by PPase for 8 h at 4° C. After cleavage and elution,mouse NMI and mouse NMI-NID were obtained (the elution volumes wereabout 30 ml respectively). Proteins were eluted with buffer contained 10mM GSH, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0. at the speed of 1 mL/minfor 30 min.

2) Size-Exclusion Chromatography

Proteins digested by PPase were further purified by Size-exclusionchromatography (HiLoad_16/60_Superdex200_prep grad and Superdex 20010/300 GL, 24 ml). The column was balanced with 150 mM NaCl, 20 mMTris-HCl, 5% gly, pH 8.0, and the flow rate was 1 ml/min eluted for 120min. The full-length mus musculus NMI protein and NMI-NID were collectedat elution volume of about 30 ml and 120 ml respectively. Finally, thepurified NMI (elution volume of 30 ml), NMI NID (elution volume of 120ml) were obtained.

The full-length and fragments of mus musculus NMI can be expressed in E.coli and purified, but no different oligomer states similar to IFP35-NIDwere observed in NMI-NID. The full-length NMI and fragments of NMIexhibited polymerized states.

Human NMI and NMI-NID were purified in the same way as mus musculus NMIrelated proteins.

This example shows that there are two oligomer states of IFP35-NID:dimer and octamer. The two NID domains in IFP35 are probably not twoindependent domains but two swapping-interacting domains. The twoaggregation states of the IFP35-NID indicated that IFP35 may have twodifferent oligomer states in cells which most likely are in accordancewith the functions. NMI had similar function with IFP35, and theirstructures may be similar. However, NMI did not have different oligomerstates when compared to IFP35. NMI secreted out from cells washomogenous polymer states. Based on estimation by the weight, thesecreted NMI was possibly a dimer or a tetramer.

In the subsequent function experiments, it was found that only octamericIFP35-NID can induce the same inflammation induced by NMI, suggestingthat the octameric IFP35-NID possessed same structural features with thefull-length IFP35. Therefore, finding amino acids on the octamericsurface which are involved in the binding of IFP35 to receptors andblocking the binding may block IFP35 from functioning normally.Similarly, inhibitory antibodies for IFP35 and/or NMI can be developedto target the amino acid residues that participate in the interaction ofIFP35 with its cellular receptors.

Example 3 Analysis of IFP35 Immune Function

IFP35 Causes Excessive Immune Response

Homo sapiens IFP35-NID1, IFP35-NID2, or IFP35-NID-H domain (dimer oroctamer) can be used as antigen to stimulate mice immune response. Fourmice were used for each protein, each mouse immune for four times, 0.2mg protein per body was injected intraperitoneally in mice. Then micestrengthen immune once every 14 days after the first time of immune, atotal of four times.

In the immune process, using Homo sapiens IFP35-NID dimer to immune micecan get corresponding antibody as expected and NID-H octamer causedswelled belly when immune to the third time and almost all the mice diedwith swelled belly when immune to the fourth time. The NID-H octamercould stimulate inflammatory response. RAW 264.7 macrophages (from ATCC)were cultured in high-glucose Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum at 37° C.

IFP35-NID1 and IFP35-NID2 had No Significant Difference.

IFP35 Immunological Function In Vivo

BMDM cell culture: Kill the mice through breaking the mice neck, takethe two hind legs of mice, soak the legs in 70% alcohol for 1 min, andtry to eliminate the muscles of the back bone, flush the marrow cavitywith PBS. Then centrifuge at 400 g for 100 min and carefully remove thesupernatant. Erythrocyte cells were collected in cell lysis buffer andthen add 10 ml PBS to neutralize the cell lysis buffer. The BMDM cellscultured in high-glucose Dulbecco's modified Eagle's medium supplementedwith 10% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamineand 20 ng/ml MCSF at 37° C.

RAW 264.7 macrophages (from ATCC) were cultured in high-glucoseDulbecco's modified Eagle's medium supplemented with 10% fetal bovineserum at 37° C.

1. Infection by Salmonella in Macrophages264.7 and BMDM Causes IFPRelease

Cells were washed three times with preheat PBS. Raw 264.7 and BMDM cellswere digested by pancreatic enzyme and counted by the hemocytometer toconfirm the number of the cells in every hole. 1 ml of preheating DMEMwas added to the cell culture dishes. Salmonella SR-11 were centrifugedat 12000 RPM for 10 min and washed twice with PBS. Salmonella weresuspended and counted by the hemocytometer. The Raw264.7 cells wereadded the Salmonella according to the multiplex of infection (1:100 or1:10), while BMDM cells according to the multiplex of infection (1:10 or1:2). Cell culture dishes were centrifuged at 1500 RPM for 10 min atroom temperature which is benefit for bacteria to adsorb on the cells.When Raw264.7 cells were infected with Salmonella for 1 hour and BMDMwere infected with Salmonella for 0.5 hours, the salmonella in the cellculture were removed and 100 μg/ml Amikacin DMEM was added in themedium. When Raw 264.7 cells were infected with Salmonella for 3 hourand BMDM cells were infected with Salmonella for 1 hour, 10 μg/mlAmikacin DMEM was added in the medium. Secretory protein in the cellculture supernatant was collected after Raw 264, 7 and BMDM cells wereinfected with Salmonella for 1, 3, 5, 9 h or 0.5, 1, 2, 4 h,respectively.

Concentrate the cell culture supernatant and extract the whole celllysates after cells were infected with Salmonella: Concentrate the cellculture supernatant: (1) The cell culture supernatant was added 0.1volumes of ice-cold 100% TCA and placed on ice for 2 h. (2) Centrifugeat 12,000 g for 30 min at 4° C. (3) Carefully remove the supernatant,wash the sediment twice with cold acetone. (4) Centrifuge at 12,000 gfor 10 min at 4° C. (5) The sediment was heated at 95° C. for 5 minutesin the 30 ul 1×SDS-PAGE loading buffer.

Extract the whole cell lysates after cells were infected withSalmonella: (1) Collect the cells: Remove the cell culture supernatant,wash the cells twice with PBS, add 1 ml PBS to the cell culture dishes,scrape the cells with cell scratcher, transfer the cell suspension to1.5 ml tube with pipet, centrifuge at 1500 RPM for 5 min and remove thesupernatant. Cell sedimentation is used to extract total protein. (2)Cell lysis: Take appropriate amount of RIPA lysis buffer, add PMSF tothe lysis buffer with a final concentration of 1 mm, proteinaseinhibitors cocktail (Roche). Add 150 ul RIPA lysis buffer to each holein the cell culture dishes and split the cells for 15 min on ice. (3)Collect the cell lysis buffer: centrifuge at 12000 RPM for 5 min at 4°C. after cells were splitted completely and transfer the supernatant toa new tube. (4) Boil the sample: The cell lysis buffer was heated at 95°C. for 5 minutes in the 150 ul 2×SDS-PAGE loading buffer.

As shown in FIG. 18, there is no or little IFP35 in the in the cellculture supernatant. However, within 1 hour after stimulated bySalmonella, released IFP35 could be detected in the culture supernatantof RAW 264.7 cells. In the following several hours, they cumulated inthe culture in time-dependent manner. In contrast, the release time ofIFP35 is earlier than the known danger signals HMGB-1. And the proteinlevel of IFP35 in cells decrease (FIG. 18A). Same results were obtainedwhen BMDM and PBDM cells were used (FIG. 18B and FIG. 18C).

To better observe the change of intracellular IFP35 protein contentafter infected with salmonella, immunofluorescence method was used todetect the IFP35 protein. As shown in FIG. 19, IFP35 was stained by redand DAPI was stained by blue. The two different schemes of infection,which is the ratio of the cells number to MOI is 1:2 or 1:10respectively, were used to observe the change of IFP35 protein contentfrom 0 to 3 hours. It was found that the intracellular IFP35 proteincontent gradually reduced as the extension of time, and the IFP35reduced faster in the higher MOI infected cells. It further proved thatthe infection induced IFP35 accumulation was not occurring inside thecells, but largely secreted to the outside of the cells. This result isin agreement with the previous research that the change of theintracellular IFP protein content.

2. The Release of IFP35 in the Virus Infection

Experimental method: The Raw264.7 cells were added the virus accordingto the multiplex of infection. Collect the cell culture supernatant intime-dependent manner. Centrifuge at 200 g for 30 min at 4° C. Collectthe supernatant by trichloroacetic acid (TCA), western blot.

Whether DNA virus (MHV68), or RNA viruses (A59) infect the Raw264.7 invitro, IFP35 release as early as 8 hours after infection when themultiplex of infection (MOI) is 0.2. Under the same conditions, HMGB1 isnot release. Increase the multiplex of infection to 5, HMGB1 releaseobviously after infection for 24 h. Similar with bacterial infectedcells in vitro, IFP35 release earlier than HMGB1, illustrated that IFP35is a very important danger signal molecules.

3. The Model of Mice Peritonitis

FIG. 21A shows that in the standard strains of salmonella SR-11 causedmice peritonitis model, IFP35 could be released. Injecting with Livesalmonella (Live SR-11) caused mice peritonitis, IFP35 could be releasedto the mice ascites. While injecting with symbiotic E. coli (E. coliStable3) and heat death of salmonella (HK SR-11, heat killed SR-11) doesnot cause inflammation and IFP35 could not be detected. The resultillustrated that IFP35 has correlation with the inflammatory response.

The establishment of the standard strains of salmonella SR-11 causedmice peritonitis model. Wash the mice ascites with 8 ml PBS after injectbacteria for 2 h, 4 h, 8 h, extract 4 ml ascites wash buffer.Trichloroacetic acid (TCA) precipitation and detect the IFP35 by westernblot. (1) C57/B6 mice, male, 8 to 10 weeks, weight about 20 g, ban waterand food for 4 hours. (2) Each mice inject 2*10̂⁵ bacteria. (3) Wash themice ascites with 8 ml PBS after inject bacteria for 2 h, 4 h, 8 h,extract 4 ml ascites wash buffer. (4) Centrifuge at 200 g for 10 min at4° C., collect the cell culture, Trichloroacetic acid (TCA)precipitation. (5) Detect the IFP35 by western blot.

As shown in FIG. 21B, when LPS, ATP activate inflammasome, IFP35 couldbe released. Experimental Method: (1) Raw264.7 cells were stimulated byLPS (1 μg/ml) for 4 h; (2) Cells were continue stimulated by ATP (5 mM)for 10, 30, 60 min; (3) Centrifuge at 200 g for 10 min at 4° C., collectthe cell culture, Trichloroacetic acid (TCA) precipitation; (4) Detectthe IFP35 by western blot.

IFP35 in Inflammatory Response

1. IFP35 induces the production of inflammatory cytokines

Experiment procedure: Wipe out the endotoxin of IFP35 dimer and octamerin the example 1, add them to the BMDM cell culture medium (finalconcentration of 5, 25, 50 μg/ml), stimulate for 4 hours and detect theknown pro-inflammatory factors, TNF-α and IL-1β using Q-PCR, GAPDH asinternal.

Q-PCR Amplification Primers:

IL-1β: Sense: 5′AAGGAGAACCAAGCAACGACAAAA3′ Antisense:5′TGGGGAACTCTGCAGACTCAAACT3′ TNF-α: Sense: 5′CCAGTGTGGGAAGCTGTCTT3′Antisense: 5′AAGCAAAAGAGGAGGCAACA3′ GAPDH: Sense:5′AGGTCGGTGTGAACGGATTTG3′ Antisense: 5′ TGTAGACCATGTAGTTGAGGTCA3′

As shown in FIG. 22, the known pro-inflammatory factors, TNF-α, IL-1β,iNOS and CD86 can be up-regulated in the presence of NID-H octamer inBMDM cells, but the NID-H dimer did not have this effect. The resultdemonstrated that different aggregation states of IFP35-NID havedifferent effect.

IFP35 NID1 and IFP35 NID2 use the same method and the results have nosignificant difference.

2. IFP35 Leads to Excessive Inflammatory Response

IFP35-NID dimer and octamer were injected into mouse peritoneal. 8 hourslater, IFP35-NID octamer caused an acute inflammatory response,manifested by neutrophil accumulation. This effect should be the reasonwhy the mice abdominal distension and death in the early inflammatoryresponse. The result demonstrated that IFP35-NID octamer not only inducethe macrophage to produce inflammation factor in vitro, but also havethe ability to regulate inflammatory responses in mice.

3. IFP35 Activates the NF-κB Pathway in Macrophages

The activation of NF-κB by IFP35-NID octamer was observed. NF-κB pathwayis an important pathway of macrophage activation. Detection method: Addendotoxin free protein of IFP35-NID dimer and octamer to the Raw264.7cells, stimulate for 15, 30, and 60 min. The cells were collected in theRIPA (Proteinase inhibitor cocktail, Roche) buffer for 30 min on ice.Centrifuge at 12,000 rpm for 10 min at 4° C. Collect the supernatant andheat at 95° C. for 10 minutes in the SDS-PAGE loading buffer. Save thesample at −70 degrees. Western blot was used to detect thephosphorylation of suppressor IκBα in the NF-κB signaling pathway.Phospho-IκBα and IκBα antibody were from CST. Results as shown in FIG.24, after adding the prokaryotic expressed different polymeric state ofendotoxin free protein, expression of the octamer of IFP35-NIDrecombined protein increased the phosphorylation of IκBα, but reducedthe total IκBα protein level. It suggested the octamer of IFP35-NIDrecombined protein could activate NF-κB signaling pathway. Takentogether, these results indicated that the octamer may induce macrophageactivation to produce a large number of inflammatory cytokines byactivating NF-κB signaling pathway, but not the dimer.

The Cell Surface Receptor of IFP35

Since IFP35 induced inflammatory response by secreted to extracellularor in body fluids, the example aims to identify the cell surfacereceptor that mediated IFP35 activity, and then to detect the biologicalfunction of TLR-Myd88 pathway in this process.

BMDM cell culture: Kill the mice through breaking the mice neck, takethe two hind legs of mice, soak the legs in 70% alcohol for 1 min, andtry to eliminate the muscles of the back bone, flush the marrow cavitywith PBS. Then centrifuge at 400 g for 100 min and carefully remove thesupernatant. Erythrocyte cells were collected in cell lysis buffer andthen add 10 ml PBS to neutralize the cell lysis buffer. The BMDM cellscultured in high-glucose Dulbecco's modified Eagle's medium supplementedwith 10% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamineand 20 ng/ml MCSF at 37° C.

IFP35 or NMI stimulation: Add IFP35-NID dimer, octamer or NMI (5, 25, or50 μg/mL) to stimulate the BMDM cells for 4 h, IL-1β, and TNF-α weredetected by Q-PCR.

Extract the cell total RNA: The total RNA was extracted from BMDM cellsusing the RNA extract Kit from KangWei Company (Cat No. CW 0597). Thetotal RNA was extracted from RAW 264.7 cells using Trizol reagent(Invitrogen).

Reverse transcription: The first strand of cDNA was synthesized from 1ug of total RNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit(Cat. No. 6210A). Real-time RT-PCR was performed using the SYBR® PremixEx Taq™ (Tli RNaseH Plus) and ROX plus Q-PCR kit (Cat. NO. RR42LR). Asshown in FIG. 25, treating Myd88 knock-out BMDM cells using IFP35-NIDprotein, the activity of NID-H were sharply reduced in these cells,IL-1β (FIG. 25A) and TNF-α (FIG. 25B) could not be induced by IFP35-NID.IFP35 likely binds to the TLR receptor in the cell surface to activeNF-κB through Myd88 dependent pathway and produce inflammation factors.

TLR-4 and TLR-9 are the two main cell surface receptor proteins whichcan induce the inflammation response. As shown in FIG. 26, TLR4 mediatethe increase of IL-1β (FIG. 26A) and TNF-α (FIG. 26B) through theIFP35-NID. Compared the NID-H octamer could up-regulated the TNF-α andIL-1β in WT, TLR9−/− and TLR4−/− mice BMDM cells, IFP35 family proteinscan also be identified by TLR4, directly or indirectly.

IFP35 Monoclonal Antibody in Treating the Diseases Caused by ExcessiveInflammatory Response Such as Sepsis

Because IFP35-NID can activate macrophages and induce inflammationresponse, IFP35 may play a role in sepsis in which IFP35 can exacerbatecytokine storm in sepsis and lead to the death of mice.

1. The Expression of IFP35 in Sepsis Mice

The establishment of LPS-induced shock model: (1) the B6/C57 mice werebanned water and food for 1 night; (2) LPS (5 mg and 10 mg per kg bodyweight) was injected intraperitoneally in mice the next day morning; (3)Take the blood using the capillary 3 hours after injection; (4) Removethe mice eye and take blood 6 hours after injection.

Detect the concentration of IFP35 in the LPS-induced shock model usingELISA. (1) Antibodies are coated: purified monoclonal antibody of IFP351D7 diluted with PBS to 2 mg/ml, 100 ml per hole in enzyme label plate,4° C., over night; (2) Wash: wash three times with PBST (0.05%Tween-20); (3) Block: PBST (0.05% Tween-20) containing 2% BSA place onshaking table to fade, room temperature, 1 h; (4) Add diluted standardand samples, 100 ul per hole, 4° C., over night; (5) Wash: wash threetimes with PBST (0.05% Tween-20), wash twice with ddH₂O. (6) Color: Add100 ul TMB to each well, avoid light, place on shaking table for 10-20min. (7) Stop the reaction: Add 50 ul 2M H₂SO₄ to each well; (8) Enzymelabel plate, 450 nm scan OD.

Quantitative detecting the IFP35 concentration in blood of sepsis micemodel shows high concentration of IFP35 in the blood of LPS-inducedsepsis mice, which reaches nanogram level (about 8 ng). But the level ofHMGB1 is pg600-800 according to the previous paper. And it depends onthe time or dose of LPS injection (for 3 hours and 6 hours, LPS dose of5 mg and 10 milligrams per kilogram of body weight).

2. Increased Survival Rate of Septic Mice by Injecting IFP35 MonoclonalAntibodies

IFP35 monoclonal antibody 1D7 was injected into mice. As shown in FIG.27, IFP35 monoclonal antibody could increase the survival rate of septicmice. The results demonstrated that IFP35 monoclonal antibody 1D7 couldprotect the mice from death effectively in bacteremic model. The medialdeath time of mice that accepted the treatment of 1D7 (n=10) was 107hours, while it was 50 hours of the control.

3. For detecting the expression of inflammation factor IL-6 in miceafter IFP35 monoclonal antibody injection, the specific method was asfollows: IL-6 ELISA kit was from BD Biosciences, the detailed operatinginformation see the specification of the product.

Results as shown in FIG. 28, for the mice with severe sepsis, the IL-6protein was highly expressed in body fluids of mice by injecting IFP35antibody (1D7) but not in non-injected.

IFP35 and Anti-Tumor Treatment

The protein level of NMI is reduced in some tumors. Since IFP35 and NMIhighlighted a variety of functions in biological progressions, such aspromoting inflammatory response of biological organisms, activatingimmune system, and promoting the release of inflammatory cytokines, theinjection of purified IFP35 or NMI into blood may activate and/orenhance anti-tumor treatments. Therefore, IFP35 or NMI protein might bea promising anti-cancer drug.

Example 4 NMI Immune Function Analysis

NMI Cause Excessive Immune Response

Full length NMI and NMI-NID used as antigen to stimulate mice immuneresponse. Four mice were used for each protein, each mouse immune forfour times, 0.2 mg protein per body was injected intraperitoneally inmice. Then mice strengthen immune once every 14 days after the firsttime of immune, a total of four times.

In the immune process, using Mouse NMI-NID to immune mice can causeswelled belly when immune to the third time and almost all the mice diedwith swelled belly when immune to the fourth time. So it is difficult toget antibodies in mice. Thus, the NID-H octamer may stimulateinflammatory response.

NMI Immunological Function Research In Vivo

1. NMI is Secreted to the Extracellular Space when Cells are Infected byBacteria

The NMI and IFP35 are homologous protein. IFP35 and NMI can assembleinto high molecular mass complex (HMMC). In this example, NMI wasdetected.

BMDM cell culture: Kill the mice through breaking the mice neck, takethe two hind legs of mice, soak the legs in 70% alcohol for 1 min, andtry to eliminate the muscles of the back bone, flush the marrow cavitywith PBS. Then centrifuge at 400 g for 100 min and carefully remove thesupernatant. Erythrocyte cells were collected in cell lysis buffer andthen add 10 ml PBS to neutralize the cell lysis buffer. The BMDM cellscultured in high-glucose Dulbecco's modified Eagle's medium supplementedwith 10% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamineand 20 ng/ml MCSF at 37° C.

RAW 264.7 macrophages (from ATCC) were cultured in high-glucoseDulbecco's modified Eagle's medium supplemented with 10% fetal bovineserum at 37° C.

2. Infection by Salmonella in THP1 Cells and Detection of the ProteinLevel of NMI in the Cell Lysate and Supernatant.

Experimental method: Collect the THP1 cells in the 15 ml tube,centrifuge at 500 g for 5 min, remove the supernatant, wash the cellswith preheat PBS, centrifuge at 500 g for 5 min, remove the supernatant.Suspend the cells with DMEM and count the cells by the hemocytometer.Add 3×10⁵ cells in every hole. Add 1 ml of preheating DMEM to the cellculture dishes. Salmonella SR-11 were centrifuged at 12000 RPM for 10min and washed twice with PBS. Salmonella were suspended and counted bythe hemocytometer. The THP1 cells were added the Salmonella according tothe multiplex of infection (1:100 or 1:10). Cell culture dishes werecentrifuged at 1500 RPM for 10 min at room temperature which is benefitfor bacteria to adsorb on the cells. When THP1 cells were infected withSalmonella for 1 hour, remove the salmonella in the cell culture and add100 μg/ml Amikacin DMEM in the medium. When THP1 cells were infectedwith Salmonella for 3 hour, add 10 μg/ml Amikacin DMEM in the medium.Secretory protein in the cell culture supernatant was collected afterTHP1 cells were infected with Salmonella for 1, 3, 5, and 9 h.

Concentrate the cell culture supernatant and extract the whole celllysates after cells were infected with Salmonella: Concentrate the cellculture supernatant: The cell culture supernatant was added 0.1 volumesof ice-cold 100% TCA and placed on ice for 2 h. Centrifuge at 12,000 gfor 30 min at 4° C. Carefully remove the supernatant, wash the sedimenttwice with cold acetone. (4) Centrifuge at 12,000 g for 10 min at 4° C.(5) The sediment was heated at 95° C. for 5 minutes in the 30 ul1×SDS-PAGE loading buffer.

Extract the whole cell lysates after cells were infected withSalmonella: (1) Collect the cells: Collect the THP1 cells in the 15 mltube, centrifuge at 500 g for 5 min, remove the supernatant, wash thecells with preheat PBS, centrifuge at 500 g for 5 min, remove thesupernatant. Cell sedimentation is used to extract total protein. (2)Cell lysis: Take appropriate amount of RIPA lysis buffer, add PMSF tothe lysis buffer with a final concentration of 1 mm, proteinaseinhibitors cocktail (Roche). Add 150 ul RIPA lysis buffer to each holein the cell culture dishes and split the cells for 15 min on ice. (3)Collect the cell lysis buffer: centrifuge at 12000 RPM for 5 min at 4°C. after cells were split completely and transfer the supernatant to anew tube. (4) Boil the sample: The cell lysis buffer was heated at 95°C. for 5 minutes in the 150 ul 2×SDS-PAGE loading buffer.

THP1 cell culture: THP1 cells were cultured in high-glucose Dulbecco'smodified Eagle's medium supplemented with 10% fetal bovine serum at 37°C.

As shown in FIG. 29, there is no or little NMI in the in the cellculture supernatant. However, within 1 hour after stimulated bySalmonella, released NMI could be detected in the culture supernatant ofTHP1 cells. Meanwhile, the NMI in the cells does not change much. Theresult demonstrated that NMI could be secreted into the extracellularwhen infected by the bacteria.

NMI in Inflammatory Response

Add the mouse full length NMI to the THP1 cell culture medium (finalconcentration of 5, 25, 50 μg/ml), stimulate for 4 hours and detect theknown pro-inflammatory factors, TNF-α and IL-1β.

Raw264.7 cell culture: Cells were cultured in high-glucose Dulbecco'smodified Eagle's medium supplemented with 10% fetal bovine serum at 37°C.

Extract the cell total RNA: The total RNA was extracted from BMDM cellsusing the RNA extract Kit from KangWei Company (Cat No. CW 0597). Thetotal RNA was extracted from RAW 264.7 cells using Trizol reagent(Invitrogen).

Reverse transcription: The first strand of cDNA was synthesized from 1ug of total RNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit(Cat. No. 6210A). Real-time RT-PCR was performed using the SYBR® PremixEx Taq™ (Tli RNaseH Plus) and ROX plus Q-PCR kit (Cat. NO. RR42LR).

As shown in FIG. 30, add control, LPS, NMI protein (5 μg/mL, 25 μg/mL or50 μg/mL), the known pro-inflammatory factors, TNF-α and IL-1β can beup-regulated in the presence of NMI in THP1 cells. The resultdemonstrated that NMI also could induce other inflammatory factorssimilar with IFP35.

NMI-NID1 and full length NMI were tested using the same method and theresults are not significantly different.

NMI Protein Aggregation State

The aggregation state of NMI that secreted by cells after stimulated bybacteria were tested. NMI can be secreted into cell culture medium whensalmonella infected the THP1 cells. As shown in FIG. 31, 10 ml cellculture medium which contains the secreted NMI concentrate to 2 ml usingMillipore concentration tube. The samples separated by chromatographicseparation using Superdex S-200 molecular sieve column. And then, theeluted samples were tested by western and the elution peak position ofNMI was tested by NMI antibody. The numbers in the figure is the volumeof the elution buffer. It shows that the top of NMI elution peak primaryat 78 ml, the corresponding molecular weight is from 70 KDa to 140 KDa.

The Cell Surface Receptor of NMI

Since NMI can be secreted out of the cell and into body fluid to induceinflammation response, this example aims to identify the cell surfacereceptor of NMI and then to detect the biological function of TLR-Myd88pathway in this process.

BMDM cell culture: Kill the mice through breaking the mice neck, takethe two hind legs of mice, soak the legs in 70% alcohol for 1 min, andtry to eliminate the muscles of the back bone, flush the marrow cavitywith PBS. Then centrifuge at 400 g for 100 min and carefully remove thesupernatant. Erythrocyte cells were collected in cell lysis buffer andthen add 10 ml PBS to neutralize the cell lysis buffer. The BMDM cellscultured in high-glucose Dulbecco's modified Eagle's medium supplementedwith 10% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamineand 20 ng/ml MCSF at 37° C.

Extract the cell total RNA: The total RNA was extracted from BMDM cellsusing the RNA extract Kit from KangWei Company (Cat No. CW 0597). Thetotal RNA was extracted from RAW 264.7 cells using Trizol reagent(Invitrogen).

Reverse transcription: The first strand of cDNA was synthesized from 1ug of total RNA using PrimeScript™ II 1st Strand cDNA Synthesis Kit(Cat. No. 6210A). Real-time RT-PCR was performed using the SYBR® PremixEx Taq™ (Tli RNaseH Plus) and ROX plus Q-PCR kit (Cat. NO. RR42LR).

NMI Monoclonal Antibody in Treating the Diseases Caused by ExcessiveInflammatory Response Such as Sepsis

Because of the high similarity of NMI structure and function with IFP35,and the cooperation of these two proteins, inhibition of NMI may havethe same effect with IFP35 to inhibit the NMI-induced overactive immuneresponse. Therefore, the NMI inhibitor is expected to treat septicemia,virus infection-induced NMI over-expression, and autoimmune diseases andso on, like IFP35 inhibitors.

The Expression of NMI in Sepsis Mice

The establishment of LPS-induced shock model: (1) the B6/C57 mice werebanned water and food for 1 night; (2) LPS (5 mg and 10 mg per kg bodyweight) was injected intraperitoneally in mice the next day morning; (3)Take the blood using the capillary 3 hours after injection; (4) Removethe mice eye and take blood 6 hours after injection.

Detect the concentration of NMI in the LPS-induced shock model usingELISA. Quantitative detecting the NMI concentration in blood of sepsismice model, it shows high concentration of NMI in the blood ofLPS-induced sepsis mice, that reaches nanogram level. But the level ofHMGB1 is pg600-800 according to the previous paper. And it depends onthe time or dose of LPS injection (for 3 hours and 6 hours, LPS dose of5 mg and 10 milligrams per kilogram of body weight). NMI monoclonalantibody can increase the survival rate of septic mice.

Overreaction for Immune Response

The different length of purified proteins that expressed by exogenousprokaryotic system, were injected into mice abdominal cavity, 100 mgprotein per mouse, after eliminating the endotoxin. The recruitment ofsome immune cells by IFP35 and NMI in mice abdominal cavity, such asneutrophils, was detected by flow cytometry.

NMI in Cancer Treatment

Based on the present disclosure of IFP and NMI, drugs can be developedto treat sepsis, inflammation storm, autoimmune diseases and otherdiseases, which are associated with abnormally high levels of IFP35and/or NMI expression, secretion, and/or activity. In some aspects,provided herein are (1) small compounds or peptides to inhibit IFP35 andNMI release; (2) antibodies to IFP35 and/or NMI to suppress thegeneration and function of IFP35 and NMI; (3) a small molecule tointerference the formation of IFP35 or NMI polymer in vivo; (4) agentsthat inhibit the secretion of interferon to reduce the generation ofIFP35; (5) agents that inhibit the interaction between IFP35 and NMIwith their cell surface receptors to suppress IFP35 and/or NMI function;(6) agents that inhibit the interaction of IFP35 and NMI with theirreceptors to suppress their function. Likewise, under the condition oflow immunity or immune suppression, such as in tumor tissues, thefunction of immune response could enhance by supply IFP35 or NMI to theorganism or a part of it.

The techniques to achieve this purpose may: (1) directly inject IFP35 orNMI protein or their targeted derivative into the organism; (2) directlyoffer a derived peptide or small molecule by IFP35 or NMI or theirtargeted derivative. In addition, in terms of diagnosis of sepsis andinflammatory response, ELISA kits can be developed. Because it is only afragment of IFP35 that expressed and crystallized in vitro, the fulllength of IFP35 may not have to form polymers to perform the function toinduct cytokine storm. In mice, using the neutralizing monoclonalantibody of IFP35 could inhibit cytokine storm. This result suggeststhat IFP35 is a factor which causes cytokine storm no matter whatpolymeric state it exists. Therefore, IFP35 might be a primary reasonthat causes cytokine storm, which makes it become a target forinhibition of cytokine storm. Thus, the proper use of IFP35 monoclonalantibody or other inhibitory molecules of IFP35 could be helpful toinhibit cytokine storm. Because of the high similarity of NMI structureand function with IFP35, and the cooperation of these two proteins thatboth secrete to the outside of the cells, and the functions like inducethe generation of cell inflammation factors, it indicate that NMI mayhave the same or similarity function with IFP35. Therefore, theinhibition of NMI could also inhibit the inflammation, and also be ableto protect animals from overreacted inflammation response. Therefore, insome aspects, death or injury induced by overproduction of NMI or NMImediated excessive inflammatory response can be avoided. Due to the lessexpression of NMI in some tumors, supplement of NMI provides ananti-tumor function. The similarity of NMI with IFP35 shows the twoaspects of function, one is to enhance the immunity of organism toinhibit cancer or avoid infection, second is that they could become thetarget of tumor treatment to curb excessive immune function byinhibiting their activity, and then to prevent and treat the organismdamage by inflammatory outbreak. Taken together, these results indicatethe crucial function to induce inflammation response by IFP35 and NMIexpression, and further demonstrate their significant value in medicalscience.

Example 5 Generation of IFP35 Antibody

To clone the sequences of the variable regions in the antibody, 7 pairsPCR primer were designed for the light chains variable region (VL),according to the 6 families of light chain. Primers P1-P7 are 7 upstreamprimers starting from FWR1 region, and P8 is the downstream primer endedat the end of FWR4 region. According to the 4 families of heavy chainsvariable regions, 5 pairs of heavy chain VH primers were designed forPCR amplification. Primers P9-P12 are the upstream primers, startingfrom FWR1 region; Primer 13 is the downstream primer ended at the end ofFWR4.

The sequences of these primers are listed below:

P1 GACATTGTGATGWCACAGTCTCC P2 GATRTTKTGATGACYCARRCTCC P3GACATTGTGCTGACCCAATCTCC P4 GACATTGTGCTGACACAGTCTCC P5SAAAWTGTKCTCACCCAGTCTCC P6 GAYATYMAGATGACMCAGWC P7 GAYATTGTGATGACMCAGWCTP8 (VL-R) TTTBAKYTCCAGCTTGGTSCC P9 AGGTGCAGCTKMAGGAGTCAGG P10AGGTYCAGCTKCARSARTCT P11 AGGTCCARCTGCAGCAGYCT P12 AGGTGMAGCTKGWGGARTCTGGP13 (VH-R) TGGTCGACGCTGAGGAGACGGT

IFP35 antibody hybridoma cells were cultured and collected, using Trizolreagents (Life Technologies Inc.) to extract total RNA. Then using theextracted RNA as template to synthesize cDNA by using cDNA synthesisreagent kit from Life Technologies Inc. The PCR fragment products oflight chain variable regions and heavy chain regions were recovered fromthe agarose gel and then cloned to T-vector (GE Healthcare). Theseconstructs were transformed to E. coli DH5α competent cells and singleclones were selected and sequenced.

Sequencing comparison results of our heavy and light chains comparingwith sequences in NCBI BLAST shows that our sequences harbor similarfeature of mouse IgG.

(SEQ ID NO: 12) GACATTGTGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACCCATCACGTTCGGTGCTGGCACCAAGCTGGAAATCAAA

Range 1: 31 to 329 GenBank Graphics Score Expect Identities Gaps Strand547 bits (296) 3e-152 298/299 (99%) 0/299 (0%) Plus/Plus Query  18CAGACTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCC  75 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct  21CAGTCTCCAGCAATCATGTCTGCATTTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCC  90 Query 76 AGCTCAAGTGTAAGTTACATGCACTGTTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGA 135|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct  91AGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGA 150 Query136 TGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGG 195|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 151TGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGG 210 Query196 TCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCGCTTAT 255|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 211TCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCGCTTAT 270 Query256 TACTGCCAGCAGTGGAGTAGTAACCCACCCATCACGTTCGGTGCTGGGACCAAGCTGGA 314||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 271TACTGCCAGCAGTGGAGTAGTAACCCACCCATCACGTTCGGTGCTGGGACCAAGCTGGA 329 ▾ NextMatch ▴ Previous Match

(SEQ ID NO: 11) TGGTCGACGCTGAGGAGACGGTGACTGAGGTTCCTTGACCCCAGTAGTCCATAGCCCAAGAGTACCCGTATCTTGCACAGAAATATGTAGCCGTGTCCTCATTCTTGAGGTTGTTGATCTGCAAATAGGCAGTGCTGGCAGAGGTTTCCAAAGAGAAGGCAAACCGTCCCTTGAAGTCATCAGCAAATGTTGGCTCTCCAGTGTAGGTGTTTATCCAGCCCATCCACTTTAAACCCTTTCCTGGAGCCTGCTTCACCCAGTTCATTCCATAGTTTGTGAAGGTATACCCAGAAGCCTTGCAGGAGATCTTGACTGTCTCTCCTGACTCCTTAAGCT GCACCT

Range 1: 8 to 331 GenBank Graphics Score Expect Identities Gaps Strand518 bits (280) 3e-143 313/328 (95%) 5/328 (1%) Plus/minus Query  10CTGAGGAGACGGTGACTGAGGTTCCTTGACCCCAGTAGTCCATAGCCC~AAGAGTACCCG  68||||||||||||||||||||||||||||||||||||||||||||||||  |  | |   | Sbjct 331CTGAGGAGACGGTGACTGAGGTTCCTTGACCCCAGTAGTCCATAGCCCTCACGG~A~~~G 276 Query 69 TATCTTGCACAGAAATATGTAGCCGTGTCCTCATTCTTGAGGTTGTTGATCTGCAAATAG 128 |||||||||||||||||||||||||||||||||| |||||||||||||||||||||||| Sbjct 275TTTCTTGCACAGAAATATGTAGCCGTGTCCTCATTTTTGAGGTTGTTGATCTGCAAATAG 216 Query129 GCAGTGCTGGCAGAGGTTTCCAAAGAGAAGGCAAACCGTCCCTTGAAGTCATCAGCAAAT 188||||||||||||||||||||||||||||||||||||||||||||||||||||||||| || Sbjct 215GCAGTGCTGGCAGAGGTTTCCAAAGAGAAGGCAAACCGTCCCTTGAAGTCATCAGCATAT 156 Query189 GTTGGCTCTCCAGTGTAGGTGTTTATCCAGCCCATCCACTTTAAACCCTTTCCTGGAGCC 248|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 155GTTGGCTCTCCAGTGTAGGTGTTTATCCAGCCCATCCACTTTAAACCCTTTCCTGGAGCC  96 Query249 TGCTTCACCCAGTTCATTCCATAGTTTGTGAAGGTATACCCAGAAGCCTTGCAGGAGATC 308|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct  95TGCTTCACCCAGTTCATTCCATAGTTTGTGAAGGTATACCCAGAAGCCTTGCAGGAGATC  36 Query309 TTGACTGTCTCTCCTGACTCCTTAAGCT 336 |||||||||||||| | || ||| |||| Sbjct 35 TTGACTGTCTCTCCAGGCTTCTTCAGCT   8 ▾ Next Match ▴ Previous Match

Translated light chain protein sequence and heavy chain protein sequencebased on the DNA sequences:

Light Chain: (SEQ ID NO: 10)D I V M T Q S P A I M S A S P G E K V T M T CS A S S S V S Y M H W Y Q Q K S G T S P K R WI Y D T S K L A S G V P A R F S G S G S G T SY S L T I S S M E A E D A A T Y Y C Q Q W S SN P P I T F G A G T K L E I K; Heavy Chain: (SEQ ID NO: 9)V Q L V E S G P E L K K P G E T V K I S C K AS G Y T F T N Y G M N W V K Q A P G K G L K WM G W I N T Y T G E P T F A D D F K G R F A FS L E T S A S T A Y L Q I N N L K N E D T A TY F C A R Y G Y S W A M D Y W G Q G T S V T V S S A S T.

1. A method for treating and/or preventing a disease or disorderassociated with abnormally high level and/or activity of IFP35(Interferon-induced Protein 35 kD) and/or NMI (N-Myc-interactingprotein) in a subject, which method comprises administering, to asubject in need of such treatment and/or prevention, an effective amountof an agent that prevents or reduces production and/or an activity ofIFP35 and/or NMI in said subject. 2-6. (canceled)
 7. The method of claim1, wherein the agent comprises a molecule, such as a small molecule or apolypeptide, that inhibits or reduces the expression and/or activity ofIFP35 and/or NMI.
 8. The method of claim 1, wherein the agent comprisesan antibody or antigen binding fragment thereof that inhibits or reducesthe expression and/or activity of IFP35 and/or NMI.
 9. The method ofclaim 1, wherein the agent comprises a polynucleotide (such as an siRNA,shRNA, or miRNA) targeting the gene encoding IFP35 and/or NMI.
 10. Themethod of claim 1, wherein the agent comprises an antisense RNAtargeting the gene encoding IFP35 and/or NMI.
 11. The method of claim 1,wherein the agent comprises a molecule that inhibits or reduces theoligomerization of IFP35 and/or NMI.
 12. The method of claim 1, whereinthe agent comprises a molecule that inhibits or reduces the expressionand/or activity of an interferon, thereby inhibiting or reducing theexpression and/or activity of IFP35 and/or NMI.
 13. The method of claim1, wherein the agent comprises a molecule that inhibits or reduces thesecretion of IFP35 and/or NMI.
 14. The method of claim 1, wherein theagent comprises a molecule that inhibits or reduces the interactionbetween IFP35 and/or NMI and a cellular receptor of IFP35 and/or NMI.15. The method of claim 1, wherein the agent comprises a molecule thatinhibits or reduces the interaction between IFP35 and/or NMI and a IFP35and/or NMI cell surface receptor. 16-17. (canceled)
 18. The method ofclaim 1, wherein the agent comprises an antibody or antigen bindingfragment that specifically binds to IFP35 and/or an antibody or antigenbinding fragment that specifically binds to NMI.
 19. The method of claim18, wherein the antibody or antigen binding fragment specifically bindsone or more NIDs (NMI/IFP35 domains). 20-24. (canceled)
 25. Apharmaceutical composition for treating and/or preventing a disease ordisorder associated with abnormally high level and/or activity of IFP35and/or NMI in a subject, which pharmaceutical composition comprises aneffective amount of an agent that prevents or reduces production and/oran activity of IFP35 and/or NMI in a subject and a pharmaceuticallyacceptable carrier or excipient. 26-28. (canceled)
 29. An antibody orantigen binding fragment thereof that specifically binds to IFP35 and/orNMI, wherein the antibody or antigen binding fragment specifically bindsto an epitope within SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ IDNO:
 8. 30. The antibody or antigen binding fragment of claim 29, whereinthe antibody or antigen binding fragment specifically binds to anepitope within: amino acids 81-170, 177-268, or 136-216 of SEQ ID NO: 2;amino acids 81-168, 175-266, or 134-214 of SEQ ID NO: 4; amino acids104-193, 202-293, or 151-250 of SEQ ID NO: 6; or amino acids 103-192,201-292, or 151-240 of SEQ ID NO:
 8. 31-33. (canceled)
 34. An antibodyor antigen binding fragment thereof that specifically binds to IFP35and/or NMI, wherein the antibody or antigen binding fragment thereofcomprises a heavy chain variable region comprising a complementaritydetermining region (CDR) consisting of the amino acid sequences of a CDRin the heavy chain variable region sequence set forth in SEQ ID NO: 9and/or a light chain variable region comprising a CDR consisting of theamino acid sequence of a CDR in the light chain variable region sequenceset forth in SEQ ID NO:
 10. 35-45. (canceled)
 46. An isolatedpolynucleotide encoding an antibody or antigen binding fragment thereofthat specifically binds to IFP35 and/or NMI, wherein the antibody orantigen binding fragment thereof comprises a heavy chain variable regioncomprising a complementarity determining region (CDR) consisting of theamino acid sequence of a CDR in the heavy chain variable region sequenceset forth in SEQ ID NO: 9 and/or a light chain variable regioncomprising a CDR consisting of the amino acid sequences of a CDR in thelight chain variable region sequence set forth in SEQ ID NO:
 10. 47-53.(canceled)
 54. An isolated polypeptide comprising, consistingessentially of, or consisting of: (1) the sequence set forth in aminoacids 81-170, 177-268, or 136-216 of SEQ ID NO: 2, optionally whereinone or more of the amino acid residues at positions 145, 147, 150, 151,172, 173, 175, 177, 182, 188, 192, 212, 199, 201, 207, 208, 210, 214,and 216 of SEQ ID NO: 2 (Ser145, Arg147, Glu150, Glu151, Asp172, Val173,Glu175, Leu177, Met182, Asp188, Gln192, Arg212, Gln199, Thr201, Gln207,Gln208, Pro210, Ser214, and Tyr216) are mutated and/or modified; or (2)the sequence set forth in amino acids 81-168, 175-266, or 134-214 of SEQID NO: 4, optionally wherein one or more of the amino acid residues atpositions 143, 145, 148, 149, 170, 172, 173, 175, 180, 186, 190, 210,197, 199, 204, 205, 206, 208, 212, and 214 of SEQ ID NO: 4 (Ser143,Arg145, Glu148, Glu149, Glu170, Arg172, Glu173, Leu175, Met180, Glu186,Gln190, Arg210, Gln197, Arg199, Arg204, Gln205, Gln206, Leu208, Ser212,and Tyr214) are mutated and/or modified; or (3) the sequence set forthin amino acids 104-193, 202-293 or 151-250 of SEQ ID NO: 6, optionallywherein one or more of the amino acid residues at positions 107, 112,117, 159, 172, 173, 192, 197, 215, 256, 267, and 292 of SEQ ID NO: 6(Leu107, Lys112, Gln117, Lys159, Glu172, Glu173, Glu192, Asp197, Asp215,Lys256, Asp267, and Glu292) are mutated and/or modified; or (4) thesequence set forth in amino acids 103-192, 201-292, or 151-240 of SEQ IDNO: 8, optionally wherein one or more of the amino acid residues atpositions 106, 111, 116, 158, 171, 172, 191, 196, 214, 255, 266, and 291of SEQ ID NO: 8 (Leu106, Lys111, Gln116, Lys158, Glu171, Asp172, Asp191,Asp196, Asp214, Arg255, Asp266, and Asp291) are mutated and/or modified.55-63. (canceled)
 64. A method for stimulating an immune response in asubject, which method comprises administering, to a subject in need ofsuch stimulation, an effective amount of the polypeptide of claim 54 insaid subject. 65-69. (canceled)
 70. An isolated polynucleotide encodinga polypeptide comprising the polypeptide of claim
 54. 71. A method ofdiagnosis, prognosis or treatment monitoring of a disease or disorderassociated with abnormally high level and/or activity of IFP35 and/orNMI in a subject, which method comprises assessing the level and/or anactivity of IFP35 and/or NMI in a subject suspected of or being treatedfor a disease or disorder associated with abnormally high level and/oractivity of IFP35 and/or NMI. 72-84. (canceled)
 85. A method ofcompanion diagnostics of a disease or disorder associated withabnormally high level and/or activity of IFP35 and/or NMI in a subject,which method comprises determining the genetic status of IFP35 and/orNMI gene in a subject being treated for the disease or disorderassociated with abnormally high level and/or activity of IFP35 and/orNMI. 86-87. (canceled)
 88. A method for identifying a modulator of IFP35and/or NMI, which method comprises: a) contacting IFP35 and/or NMI witha test substance and assessing an activity of IFP35 and/or NMI that hasbeen contacted by said test substance; b) assessing an activity of saidIFP35 and/or NMI that has not been contacted by said test substance; andc) comparing said activities of IFP35 and/or NMI assessed in steps a)and b), and identifying said test substance as a modulator of IFP35and/or NMI when said activities of IFP35 and/or NMI assessed in steps a)and b) are different. 89-93. (canceled)